*Review Curcuma Longa***, the "Golden Spice" to Counteract Neuroinflammaging and Cognitive Decline—What Have We Learned and What Needs to Be Done**

**Alessandra Berry 1,\*,†, Barbara Collacchi 1,†, Roberta Masella 2, Rosaria Varì <sup>2</sup> and Francesca Cirulli 1,\***


**Abstract:** Due to the global increase in lifespan, the proportion of people showing cognitive impairment is expected to grow exponentially. As target-specific drugs capable of tackling dementia are lagging behind, the focus of preclinical and clinical research has recently shifted towards natural products. Curcumin, one of the best investigated botanical constituents in the biomedical literature, has been receiving increased interest due to its unique molecular structure, which targets inflammatory and antioxidant pathways. These pathways have been shown to be critical for neurodegenerative disorders such as Alzheimer's disease and more in general for cognitive decline. Despite the substantial preclinical literature on the potential biomedical effects of curcumin, its relatively low bioavailability, poor water solubility and rapid metabolism/excretion have hampered clinical trials, resulting in mixed and inconclusive findings. In this review, we highlight current knowledge on the potential effects of this natural compound on cognition. Furthermore, we focus on new strategies to overcome current limitations in its use and improve its efficacy, with attention also on gender-driven differences.

**Keywords:** turmeric; aging; brain; cognition; bioavailability; oxidative stress; inflammation

#### **1. Introduction**

Cognitive decline is a highly disabling and prevalent condition in the aging population, greatly affecting physical health and quality of life. Global average life expectancy, as observed in 2019 by the Global Health Observatory (GHO), was estimated to be 73.4 years in the WHO European Region (https://www.who.int/gho/mortality\_burden\_disease/ life\_tables/situation\_trends\_text/en/ accessed on 25 April 2021). In 2050, the number of people over the age of 60 is expected to reach a total of about 2.1 billion (https:// www.who.int/ageing/publications/active\_ageing/en/ accessed on 25 April 2021). As the ageing population is rapidly growing due to the global increase in life expectancy in westernized life-style countries, the number of people experiencing cognitive impairment is also expected to grow in parallel. Disregarding overt pathologies, the impact of age itself on cognitive abilities is so disruptive and so underestimated that it has been described as "the elephant in the room" [1,2].

The lack of effective pharmacotherapy has led researchers to seek alternative approaches in order to treat or prevent the cognitive decline accompanying ageing. Accumulating evidence suggests that conditions co-occurring in metabolic dysfunctions such as neuroinflammation, oxidative stress (OS), mitochondrial dysfunction or autophagy may all potentially act as triggers for cognitive decline. Indeed, metabolic syndrome (MetS, defined as the presence of three or more of the following five medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides (TG) and low serum

**Citation:** Berry, A.; Collacchi, B.; Masella, R.; Varì, R.; Cirulli, F. *Curcuma Longa*, the "Golden Spice" to Counteract Neuroinflammaging and Cognitive Decline—What Have We Learned and What Needs to Be Done. *Nutrients* **2021**, *13*, 1519. https:// doi.org/10.3390/nu13051519

Academic Editor: Giuseppe Grosso

Received: 31 March 2021 Accepted: 26 April 2021 Published: 30 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

high-density lipoprotein—HDL), negatively impacts cognitive performance and brain function possibly increasing neuroinflammation, OS and brain lipid metabolism [3]. Insulin resistance (IR, defined as the inability of peripheral target tissues to respond normally to insulin) is a common condition experienced at old age and often associated with obesity. It typically precedes the onset of type 2 diabetes (T2D) by several years and is considered as a risk factor for cognitive decline in both diabetic and non-diabetic populations [4]. In fact, peripheral IR, by decreasing insulin signaling within the brain, may alter its metabolic functions, increasing OS and neuroinflammation, eventually setting the stage for dementia and neurodegeneration. Thus, neuroinflammation, OS and metabolic dysfunctions involve a strict connection between the brain and the overall metabolic regulation that occur in the periphery. Moreover, changes in microbiota composition and dysbiosis, can potentially influence a number of pathological conditions, include MetS, obesity, T2D, heart failure, and cognitive function (see [5] and references therein).

While novel target-specific drugs are currently lacking [6], some epidemiological studies indicate that natural antioxidant agents, such as polyphenols, polyunsaturated n-3 fatty acids or vitamin-rich foods may delay the occurrence of neurodegenerative disorders. Polyphenols, in particular (e.g., curcumin and resveratrol) having pleiotropic protective effects appear ideal to prevent or treat conditions (such as AD) whose origin is multifactorial [7]. A growing body of research suggests that regular consumption of natural products (vegetables, fruits, leaves, roots, seeds, berries etc.) rich in polyphenols might improve health outcomes through different mechanisms boosting the organisms' antioxidant defenses [8]. Natural compounds represent a major source for the discovery of drug targets and are ever increasingly attracting the interest of the scientific community, with the main aim of validating their efficacy for the prevention and treatment of different conditions, including cognitive decline and metabolic disorders [9]. Notwithstanding the growing interest in this class of compounds, rigorous clinic trials addressing their specific effects are lacking or show biases due to the nutritional status of the subjects, genetic background, gender, treatment duration and dose–response relationship [10]. With regard to this latter point, a major drawback is related to their bioavailability, i.e., the amount of compound (or of its active principles) that reaches systemic circulation due to intestinal endothelium absorption and first-pass metabolism. Thus, the use of natural products and nutraceuticals poses important questions regarding human safety and calls for a better understanding of their therapeutic efficacy as well as their mechanisms of action.

Curcumin, one of the best investigated botanical constituents in the biomedical literature, has been receiving increased interest due to its unique molecular structure, which targets inflammatory and antioxidant pathways, and its potential to improve healthspan [11–15]. The genus Curcuma includes approximately 80 species and is regarded as one of the largest genera of the Zingiberaceae family [16]. Curcumin (1,7 bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a lipophilic polyphenol active extract deriving from the rhizome of *Curcuma longa*. Curcumin (and curcuminoid analogs such as demethoxycurcumin and bisdemethoxycurcumin) provides the characteristic bright yellowish/golden pigment of turmeric widely used in traditional Indian and Chinese medicine from thousands of years because of a number of beneficial effects on human health [17,18]. Today curcumin is used all over the world as a supplement, spice and food additive. It is considered a safe compound suitable for daily dietary use by the United States Food and Drug Administration (FDA), the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the European Food Safety Authority (EFSA) who have indicated 0–3 mg/kg as an acceptable daily intake (https: //www.fda.gov/food/generally-recognized-safe-gras/gras-notice-inventory accessed on 25 April 2021) [19]. Many of its medical uses have been mechanistically validated in in vitro and in vivo preclinical studies (more than 3000 investigations, see [20]) mainly focusing on its antioxidant and anti-inflammatory properties. In recent years, the positive effects of curcumin have been observed in several chronic diseases ranging from cardiovascular, gastrointestinal, neurological disorders and diabetes to several types of cancer [21–24]. The

consumption of curcumin has been associated with a global improvement in the glycemic and lipid profile in patients with MetS [25]. In addition, amelioration in cognitive function in animal models has been widely documented due to its action on structure and functionality of neuronal membranes [26,27]. Despite this evidence it is worth mentioning that curcumin is characterized by poor stability, a feature resulting in an overall low oral absorption, though, once in the blood stream, curcumin appears to be stable and able to reach target tissues [28]. However, as far as the brain is concerned, its application raises the critical issue of its ability to cross the blood–brain barrier (BBB), an issue deserving further investigation [29].

This paper will focus on *Curcuma longa* as a very promising natural compound to counteract inflammaging and cognitive decline. It will review its possible mechanisms of actions and efficacy and will critically address the issue of its bioavailability and describe recent strategies aimed at improving its supplementation.

#### **2. Curcumin, Cognitive Decline and Glucose Homeostasis (Peripheral and Central Actions)**

The concept of energy homeostasis is receiving much attention nowadays due to the global spread of obesity and diabetes. Hyperglycemia is one of the conditions characterizing MetS, a main risk factor for multi-cause morbidity that includes T2D, cardiovascular disease and also dementia and Alzheimer's disease (AD). Insulin plays an important role in neuronal survival, in the protection of excitatory synapses and the formation of dendritic spines through the activation of AKT, mTOR and Ras-related pathways that are part of the insulin signaling cascade (see [4] and references therein). Moreover, it regulates levels of GABA, NMDA and AMPA-mediated mechanisms involved in brain plasticity. Insulin binding is highest in the cerebral cortex that plays a role in the control of executive functions as well as in the hippocampus, a brain area involved in learning and memory [4]. The mammalian brain is a highly demanding organ in terms of energy expenditure and shows reduced capacity for cellular regeneration and poor antioxidant defenses, making it particularly susceptible to metabolic and OS insults [30]. Indeed, exposure to hypercaloric diets and obesity has been shown to decrease insulin transport into the mammalian brain, a condition that is restored upon caloric restriction (see [31] and references therein). Recent evidence suggests that IR within the brain, a condition that may be described as the inability of brain cells to respond to insulin and its receptors, has major potential to impact cognitive functions and to contribute to the etiopathogenesis of AD. IR in peripheral tissues and organs (a conditions underlying hyperglycemia and diabetes) is often associated with IR within the brain leading to insulin deficiency and impaired glucose transport inside the neurons [32]. Such a condition may lead to neuronal death, apoptosis and degeneration, predisposing the individual to neurodegenerative diseases and the resultant cognitive decline. Thus, brain desensitization to insulin receptor due to untreated T2D, obesity or chronic consumption of hypercaloric diets may play a key role in what has now been defined as a novel form of diabetes (type 3 diabetes, T3DM) and its complications [33], indicating glucose homeostasis as key in the maintenance of cognitive function.

A growing body of clinical and preclinical data suggests that curcumin holds potential for the control of glucose homeostasis [34] since it may improve glucose uptake, insulin sensitivity and beta islet cell function. Moreover, curcumin may reduce glucose and lipid levels in addition to reducing OS and inflammation [35] by interacting with almost all the players involved in these processes, as demonstrated in in vitro studies [11,35]. A very large amount of literature is now available on curcumin effects. Thus, we will only summarize some of the main aspects.

Preclinical animal models have clearly shown a main effect of natural compounds, including curcumin, on cognitive function during ageing [26,36–39]. These effects are most likely related to the ability of curcumin to act directly on Aβ plaques as well as to its anti-inflammatory and antioxidant properties. Indeed, a number of preclinical studies have reported downregulation of biomarkers of inflammation (e.g., TNF-α, IL-1β) and OS (e.g., lipid peroxidation, reactive oxygen species—ROS—nitrite and glutathione) believed

to be involved in cognitive impairments, confirming the anti-inflammatory and antioxidant properties of curcumin [40–50].

Results of published clinical studies, although in some cases not conclusive, show promise for curcumin's use as a therapeutic for cognitive decline [51]. The efficacy of curcumin supplementation in humans has been evaluated in several randomized controlled trials, suggesting its potential to reduce blood glucose, C-peptide, glycated hemoglobin (HbA1c), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in patients with T2D [52]. A recent meta-analysis provides evidence for curcumin's ability to reduce body mass index, body weight, body fat and leptin values and to increase adiponectin levels in patients with MetS and related disorders [53]. Its effectiveness in reducing TG and C-reactive protein (CRP) and increased adiponectin levels has also been reported [54]. In addition to the ability of curcumin to control glucose homeostasis, and indirectly, to improve/counteract cognitive decline, direct effects have also been observed (and are currently being studied) in the brain. In fact, curcumin has received increased interest due to its unique molecular structure that targets directly amyloid aggregation, one of the major hallmarks of AD. To this regard, Yang and colleagues have provided in vitro and in vivo evidence for curcumin to inhibit Aβ aggregation as well as to prevent oligomer formation [55]; Garcia-Alloza, in a mouse model of AD, found that curcumin reversed existing amyloid pathology and improved the associated neurotoxicity [56].

Beyond its use as a compound to prevent/counteract cognitive decline, intriguing evidence supports a possible application for in vivo diagnostics for AD and other related neurodegenerative pathologies [57]. In fact, AD diagnosis can be currently made only by means of clinical criteria supported by invasive and time consuming investigations [58]. Thus, a patient-friendly and repeatable amyloid or Aβ biomarker may alleviate the burden related to currently available diagnostic tools as well as support therapy monitoring in clinical trials [57]. In fact, den Haan and colleagues, by taking advantage of the peculiar feature of curcumin of being naturally fluorescent as well as its Aβ-binding properties, have provided evidence of a selective binding (of curcumin and its related isoforms) to fibrillar Aβ in plaques and cerebral amyloid angiopathy in post mortem AD brain tissues. However, in order to use curcumin as a feasible tool for in vivo detection of Aβ, its poor bioavailability and in vivo metabolism should be carefully considered (see below).

#### **3. Curcumin, Oxidative Stress and Inflammation**

The neuropathological features of the brain affected by dementia suggest that the oxidative and inflammatory burden plays a role in the progression of pathological signs by reducing brain plasticity, thus, being an important risk factor for cognitive disability [58]. To this regard, metabolic dysfunctions that are often associated with OS and inflammation, may greatly accelerate the onset and worsen the progression of cognitive functions by promoting brain ageing and reducing healthspan [59].

Neuroinflammatory processes are a main feature of neurodegenerative disorders in which microglia and astrocytes are over-activated, resulting in increased production of pro-inflammatory cytokines. Moreover, deficiencies in the anti-inflammatory response may also contribute to neuroinflammation. More specifically, the activated neuroglia by increasing both NF-κB, COX2 and iNOS levels may induce, in turn, the release of proinflammatory cytokines, such as IL-6, IL-1α and TNF-β. This pervasive inflammatory condition results in an overall increase in the OS burden leading to neuronal toxicity and the subsequent cognitive deficits characterizing neurodegenerative diseases. Numerous studies have indicated that curcumin is an effective antioxidant both in vivo and in vitro [9]. Curcumin treatment could attenuate cell apoptosis, decrease the level of lipid peroxidation, and increase the activity of various antioxidant enzymes including superoxide dismutase (MnSOD) and glutathione (GSH) [60], thus helping to break the vicious cycle sustaining neuroinflammation and containing the progression of neurodegenerative diseases [61]. The underlying mechanism is possibly associated with the function of NFE2-related factor-2 (Nrf2), a transcription factor promoting the upregulation of antioxidant defenses [61].

As far as apoptosis is concerned, many mechanisms have been proposed. Xi-Xun Du and colleagues indicated that curcumin's property of iron chelation and reduction may underlie its anti-apoptotic effects [62]. Chen and co-workers reported that curcumin may exert its cytoprotective effects against neurotoxic agents via its antiapoptotic and antioxidant properties through the Bcl-2–mitochondrion–ROS–inducible nitric oxide synthase pathway [63]. Moreover, Yu and colleagues reported that the inhibition of JNK pathway and the activation of caspase-3 cleavage might prevent neuronal death [64]. Indeed, the anti-inflammatory and antioxidant properties of curcumin are strictly related to its action on apoptotic pathways and on neuronal death. In fact, pro-inflammatory cytokines are not only involved in the so-called neuroinflammaging but may also trigger the apoptotic process. Likewise, excessive OS may directly lead to mitochondrial swelling and apoptosis. Thus, inflammation and apoptosis are related in a vicious cycle leading to neuronal death [65].

Recently, several studies have highlighted the role of inflammatory pathways mediated by the inflammasome in neurodegenerative diseases. In particular, the NOD-like receptor pyrin domain-containing-3 (NLRP3) has been suggested to play a pathogenic role in several neuroinflammatory diseases, including AD [66]. In vitro and in vivo studies have shown that Aβ peptide activates NLRP3 inflammasome in microglial cells. Furthermore, in a mouse model of AD, NLRP3 knockout (KO) mice were protected from impaired spatial memory performance and showed a decrease in the Aβ plaque load [67], similar results were obtained when a specific NLRP3 inhibitor was administered to mice [68]. This evidence points to the inflammasome as a potential therapeutic target for AD treatment [69]. Notably, recent evidence shows that curcumin, by modulating the activity of NLRP3 inflammasome, could be beneficial in reducing neuroinflammation and/or neurodegeneration in different neurological disorders, such as major depression, brain ischemia, AD and epilepsy [13,70,71].

OS is a condition characterizing aerobic biological systems, the major portion of ROS being generated as a by-product of the electron transport chain operating in the mitochondria [30]. As already mentioned, OS is a condition strongly associated to inflammation that may act both as (con)cause and effect of pathological conditions affecting brain ageing; however, a growing body of evidence suggests that ROS are not only responsible for oxidative damage to cells and macromolecules but they may also play a role as mediators in specific signaling cascades. Hydrogen peroxide (H2O2) in particular has been identified as a ROS able to affect the ageing process by specifically mediating insulin signaling and promoting fat accumulation, ultimately affecting the ageing process [72]. Worth to notice, the master regulator of this process is the p66Shc *gerontogene*, which, by acting within the mitochondrion, increases the generation of H2O2, amplifying insulin signaling [73–75]. Interestingly, deletion of p66Shc gene in mice resulted in the decreased formation of mitochondrial H2O2 [75], a feature that has been associated with reduced fat accumulation as well as decreased incidence of metabolic and cardiovascular pathologies [73,76]. Moreover, p66Shc KO mice were characterized by elevated resistance to OS, delayed brain ageing and improved overall healthspan, all features associated with increased brain and behavioral plasticity. In fact, the brain of p66Shc KO mice was characterized by reduced levels of inflammation and OS and increased levels of the neurotrophin brain-derived neurotrophic factor (BDNF); in addition, these mice showed decreased emotionality and improved cognitive function [77–79].

Lifestyles have the potential to modulate healthspan during ageing. For example, physical exercise and diet, as well as the consumption of nutraceutical compounds (including curcumin), may greatly contribute to reducing neuroinflammaging by targeting brain pathways related to OS and inflammation (see below, next paragraph). Physical exercise in elderly women has been shown to improve metabolic functions and this was paralleled by a decrease in the peripheral levels of p66Shc gene [80,81]. Very recently, a role for curcumin was also reported in the modulation of the p66Shc gene as its was able to downregulate the expression levels of this gene in peripheral blood mononuclear cells (PBMC), improving

diabetic nephropathy in a rat model [82]. These data overall suggest that p66Shc might be exploited as a suitable biomarker of curcumin efficacy to counteract the ageing-related burden and to improve overall healthspan.

#### **4. A Potential Mechanism of Action: Curcumin as a "Hormetin"**

A large epidemiological Indo-US Cross National Dementia study showed that rural Indian populations have a low prevalence of AD and AD-associated dementia compared to the US population, and this may be linked to the high curcumin consumption [17], although such correlation does not necessarily imply causative connection.

An ever-increasing body of evidence suggests that natural compounds may alleviate the burden of chronic diseases by increasing individuals' ability to cope with OS. Notably, differently from the consumption/administration of antioxidants (of natural or synthetic origin), whose beneficial effects are still debated, most natural compounds do not act solely as free radical scavengers but rather, and most interestingly, as "antioxidant boosters" [83]. In this regard, it is important to point out that ROS also function as signaling molecules underlying physiological processes and, for this reason, their generation and scavenging needs to be tightly regulated (see below).

As reviewed by Lee and colleagues [8], cellular stressors that are relevant to the pathogenesis of chronic diseases may be roughly categorized into four general types: (1) OS resulting from the unbalance between ROS production and the organism's antioxidant defenses; (2) metabolic stress deriving from impaired cellular bioenergetics and mitochondrial dysfunctions; (3) proteotoxic stress protein misfolding and aggregates accumulation, and (4) inflammatory stress that leads to the production of ROS from immune cells. All these stressors are relevant for brain ageing. Moreover, and most importantly, OS is also involved as a cause or as a consequence in all the above-mentioned categories. Cytotoxic effects of ROS contribute to the death of neurons during chronic neurodegenerative diseases such as AD and Parkinson's disease and also during the ageing process. However, ROS also function as signaling molecules underlying physiological processes including cell proliferation, migration, and survival through the regulation of neurotrophic factors [84,85]. Therefore, the generation and scavenging of ROS needs to be tightly regulated and nutraceutical compounds appear to be good candidates to play a role in this process. In this regard, the cells throughout the body and brain might trigger stress signaling pathways, eventually leading to the enhancement of their own resistance to further stressors, including OS. An intriguing hypothesis suggests that nutraceuticals might be perceived as potentially toxic by the organism (at high doses). However, exposure to low doses of these compounds might stimulate the organism's hormetic/adaptive responses aimed at counteracting such threats ("hormesis hypothesis", see [8]. Indeed, a recent paper by Calabrese and co-workers provides evidence that curcumin displays hormetic-like biphasic dose response features that are independent from the biological model used for investigation, cell type, and endpoints [86]. These findings hold major implications for study design, including selection of doses and sample size, also considering the specific context of bioavailability and pharmacokinetics, see Figure 1.

**Figure 1.** An intriguing hypothesis suggests that nutraceuticals might be perceived as potentially toxicant by the organism (at high doses), however, exposure to low doses of these compounds might stimulate the organism's hormetic/adaptive responses aimed at counteracting such threats. Likewise, *Curcuma longa* acting as a mild stress might trigger signaling cascades boosting antioxidant and anti-inflammatory pathways (related to Nrf2, NF-kB, NLRP3), leading to decreased neuroinflammation, increased neurogenesis and brain plasticity, finally improving cognitive abilities. Figure adapted from [87], Copyright © 2014 University of Massachusetts.

#### **5. Criticisms to Be Considered in Curcumin Supplementation**

Despite plenty of data on the positive health effects of curcumin [88,89], some problems strongly limit its effectiveness and usefulness. First of all, its low bioavailability. Curcumin is characterized by low water solubility and high instability in most body fluids; in addition, it is poorly absorbed by the gastrointestinal tract. In fact, curcumin is rapidly metabolized by the large intestine and by liver enzymes, leading ultimately to the production of sulphate and glucuronide O-conjugated metabolites [90]. Specifically, curcumin that does reach the blood flow undergoes phase I (reduction) and phase II metabolism (conjugation). Reductases reduce curcumin to dihydrocurcumin, tetrahydrocurcumin and hexahydrocurcumin (phase I) [91,92], then these phase I metabolites are conjugated to sulphates and glucuronides (phase II) [93,94].

In animal and human studies, a low concentration of curcumin in blood plasma and urine was observed after oral administration, in particular, serum curcumin levels are undetectable in humans even after high oral doses (up to 8 g/day) [95,96]. Finally, the presence of curcumin in the blood is not sufficient to ensure the delivery in the brain to exert the neuroprotective activity because several studies have demonstrated that it does not easily cross the BBB. All these factors together have pushed research towards finding new formulations or new ways of administration able to stabilize the molecule and increase its bioavailability, by reducing its metabolism and increasing the retention time in the bloodstream [97]. To this regard, the simultaneous administration of curcumin with piperine [98,99], essential oil [100] or milk [101] have been suggested to stimulate the gastrointestinal system, prevent the efflux of curcumin and to increase absorption and metabolism. Many studies have been aimed at devising and testing new drug delivery strategies using, for example, carriers such as soy lecithin phosphatidylcholine (phytosome, Meriva®) that improve both the absorption of curcumin in the intestine as well as its penetration into the cells [102–105]. In addition, nanoparticles, liposomes, micelles, phospholipid complexes, emulsions, microemulsions, nanoemulsions, solid lipid nanoparticles, nanostructured lipid carriers, biopolymer nanoparticles and microgels are able to increase curcumin bioavailability by enhancing small intestine permeation, preventing

possible degradation in the microenvironment, eventually increasing plasma half-life and enhancing curcumin efficacy [106–113]. Another way to administer curcumin is by using exosomes, nanovesicles (30–100 nm) that are generated within the cell in the endosomal network. This method, which appears to be safe and non-cytotoxic [114,115], increases both the plasma concentration as well as the bioavailability of curcumin 5–10 times more than curcumin alone [116]. Other studies have confirmed that increased solubility, stability and bioavailability of curcumin can be obtained by incorporating it into exosomes. All these different curcumin formulations enhance its bioavailability and allow for greater persistence in the body, better permeability and resistance to metabolic processes and a higher efficacy [97]. Other studies have focused on changing the chemical structure of curcumin, generating curcumin derivatives that may show not only an improved pharmacological activity, but also better physicochemical and pharmacokinetic properties [117,118], see Figure 2.

**Figure 2.** New delivery systems such as polymeric micelles, polymer nanoparticles, nanogels, dendrimers, nanoemul-sions, inclusion complexes, phytosomes, solid-lipid nanoparticles, curcumin nanoparticles and liposomesnanoparticles, liposomes, micelles, nanogel, dendrimers, nanoemulsions, inclusion complexes and phytosomes have the potential to reduce intestinal degradation and increase curcumin bioavailability, ultimately enhancing its efficacy throughout the body and the brain. Within this latter organ the increased curcumin bioavailability might counteract cognitive decline. Figure adapted with permission from [119]. Copyright © 2018, Said Moselhi.

It is worth noting that the paradox of the curcumin pharmacological effect, despite its poor bioavailability could be, at least partially, explained by the influence of the microbiome on curcumin metabolism. Indeed, the biological activities of curcumin are linked to the digestion by the intestinal flora, which can produce active metabolites. Thus, the beneficial effects of curcumin seem to depend on the individual ability to metabolize it, that is, to the composition of each person's intestinal microbiota [120]. Several microorganisms capable of modifying curcumin have been identified in the human microbiota, including *Bifidobacteria longum*, *Bifidobacteria pseudocatenulaum*, *Enterococcus faecalis*, *Lactobacillus acidophilus* and *Lactobacillus casei* [121]. Many of them metabolize curcumin to a large extent (more than 50%) and produce a number of metabolites (approximately 23 of them have been identified) by different metabolic pathways such as acetylation, hydroxylation, reduction, demethylation [122]. Several studies have shown that the metabolites have similar properties to curcumin [123] and many of them have been shown to exhibit neuroprotective effects [124,125], suggesting that curcumin transformed by the gut microbiota could be useful for microbiome-targeting therapies for AD. In fact, bidirectional communication exists between the central nervous system and the gut microbiota, which plays a key role

in human health [5]. A growing body of evidence suggests that the gut microbiota can influence human brain and behavior, and the different metabolites secreted by the gut microbiota can affect the cognitive abilities of patients diagnosed with neurodegenerative disorders [126,127]. Changes in the gut microbiota composition, caused by dietary habits, antibiotic exposure and/or infections might result in conditions of dysbiosis (also known as dysbacteriosis) that are involved in the etiopathogenesis of different diseases in humans, including MetS, obesity, T2D and neurodegenerative disorders [127,128]. Indeed, changes in gut microbiota homeostasis leads to increased intestinal permeability that, in turn, results in the translocation of bacteria and endotoxins across the epithelial barrier, a condition that might trigger an immunological response associated with the production of pro-inflammatory cytokines. Such mechanisms have the potential to greatly contribute to neuroinflammation through the secretion of pro-inflammatory cytokines, ultimately altering brain functions [5]. Thus, curcumin supplementation may be useful to counteract/prevent cognitive decline also by mechanisms involving the preservation of individuals' microbiota homeostasis.

#### **6. What have We Learned and What Needs to Be Done**

Although only a few clinical studies have examined curcumin's effect on human cognitive functioning, the results of these trials are sometimes inconsistent, highlighting the difficulty in translating basic research to the clinic. While some studies report no cognitive enhancing effects of curcumin [129,130], other data indicate positive effects of this compound on cognitive function [131–133]. Also in preclinical studies, we found great heterogeneity and limited ability to standardize age of administration or endpoints [39,73,134,135].

Nonetheless, even in very different animal models, curcumin consistently decreases both systemic and central neuroinflammation while improving redox state [51]. As many clinical studies did not include inflammatory and oxidative biomarkers, future trials should target these measures to yield further insight into why curcumin has not shown consistent cognitive effects in humans. Among these, changes in p66Shc transcription in PBMC might be exploited as a potential biomarker of curcumin efficacy [82,136]. Bioavailability may be one main factor that increases variability between studies. Administration of any of the three constituents (curcumin, bisdemethoxycurcumin and demethoxycurcumin) separately instead of the parent curcuminoid mixture was recommended as a more efficient way of treatment [125]. Furthermore, a synergistic effect of curcumin with other dietary supplements, such as piperine, α-lipoic acid, N-acetylcysteine, B vitamins, vitamin C, and folate can improve its effects [137,138]. Nowadays, nanoparticles are mainly used since they demonstrate better penetration at the BBB and cause more evident biochemical changes than free curcumin [9,46,49,139,140].

Nevertheless, there is still room for improvement. As an example, use of soy lecithin phosphatidylcholine (phytosome, Meriva®), which improves both the absorption of curcumin in the intestine as well as its penetration into the cells, has been used and has resulted in significant effects in clinical studies assessing a number of variables such as liver function, inflammation, gastrointestinal disturbances and T2D [141–145]. Thus, using new delivery systems that increase bioavailability appears to be a *must-do* for future clinical trials [26,51].

Other drawbacks of previous clinical trials are limited power, different formulations of curcumin used [129] and differences in ethnicity, as an example Caucasians vs. Asians, making it difficult to compare results derived from different studies [146]. Genetic factors, such as polymorphisms or dietary differences, such as higher consumption of curcumin in Asia, could underlie differential effects in different ethnic groups.

A dose–response relationship should also be taken into account. The optimal dose would have maximum cognitive enhancing effects with the safest pharmacokinetic profile. We have previously shown in animal studies that, in general, beneficial effects of natural compounds on cognition are dose-dependent with the higher dosages generally being more effective compared to lower dosages, although a significant effect of nutraceutical

compounds on memory retention and OS was also demonstrated at the lowest dose [39]. In addition, when selecting the most effective dose in basic or clinical studies it should be carefully taken into account that curcumin commonly displays a biphasic dose–response curve, such as hormetic compounds do [86,147]. In this regard, it should be noted that problems such as optimal dose and bioavailability are common to different natural compounds. A recent review by Mazzanti and Di Giacomo pointed out that such issues may also represent a major drawback when trying to compare the efficacy of curcuma and resveratrol, two polyphenols with very similar antioxidant and anti-inflammatory properties, to counteract cognitive decline [7]. As an example, both curcumin and resveratrol are able to activate the Nrf2 and NF-kB pathways as well as to modify insulin signaling. Their effects mostly overlap and both have been shown to enhance cognitive function in animal models [8]. More recently, Saleh and co-workers when trying to compare the protective effects of curcumin, resveratrol and sulphoraphane in an in vitro study, confirmed that enhancing the delivery of phytochemicals (either by designing novel nanostructures or using mixtures of natural compounds that work synergistically) is a priority in this field of research [148]. Thus, further research is mandatory to establish the most effective substance to be used, in which conditions and for whom.

#### **7. Targeting Both Genders**

The male-female health-survival paradox, also known as the morbidity-mortality paradox or gender paradox, poses that women live longer, though they experience more disabilities and medical issues throughout life, compared to men. Such a paradox holds true in almost every country in the world since virtually all the primary causes of death are higher for men at all ages [149]. Thus, due to their greater resilience to stress, women live longer than men but experience higher rates of physical illness, leading to debilitating, though rarely lethal, conditions. Several hypotheses have been proposed for this phenomenon that could be interpreted as a sex-driven resilience to stressors (leading to longer lifespan), including more efficient female immune functioning, the protective role of estrogens as well as increased antioxidant capacity [149].

Many chronic conditions, including dementia and AD, though not lethal *per se*, are strongly linked to disability and loss of physiological functions. Alzheimer's disease is a multifactorial neurodegenerative disorder, the development of which depends upon both environmental as well as genetic risk factors. A recent review by Christensen and Pike put the attention specifically on two risk factors that may play a key role in the initiation and/or progression of AD and that may be particularly problematic for women: inflammation and obesity [150]. Alzheimer's disease and other dementias are highly prevalent among women [151] and the onset of menopause, which is associated with estrogen breakdown, decreases women's vulnerability threshold to both metabolic and cognitive disorders that depend upon central adiposity and overall inflammation [150].

In women, the shift from adulthood to middle-age is characterized by an overall increase in the proportions of overweight and/or obese subjects [152]. Clinical and preclinical studies suggest that the age-related loss of ovarian hormones results in weight gain and contributes to changes in the distribution of adipose tissue leading to increased waist-to-hip ratio [153]. Elevated adiposity increases the risk of different pathological or sub-pathological conditions, including MetS [154], T2D [155] and AD [156]. One consequence of increased adiposity that may underlie its pathogenic role is chronic inflammation, which is observed both systemically as well as in the brain (see [150] and references therein). A large body of data supports the link between estrogens (and other sex steroid hormones) and the modulation of the individual inflammatory profile. Indeed, estrogens are powerful anti-inflammatory mediators, thus, the drop experienced by women at menopause triggers a rise in pro-inflammatory cytokines that may place tissues throughout the body at increased risk of inflammaging-associated diseases [157]. A recent clinical trial aimed at assessing the efficacy of oral curcumin (500 mg) administration twice/day for 8 weeks on anxiety and other specific symptoms that accompany menopause, has shown that curcumin

significantly reduced hot flashes in postmenopausal women [158]. In this context, it is possible to foresee the use of curcumin as a possible preventive strategy to be administered in pre-menopausal women aimed at boosting anti-inflammatory and antioxidant capacity when natural defenses that women are endowed with start to be threatened by the absence of estrogens (see Figure 3). In fact, it is important to stress here that, given the current knowledge on age-associated cognitive decline, it would be unrealistic to foresee a therapeutic use of nutraceuticals in overt pathologies characterized by massive neurodegeneration, such as AD. Thus, early diagnosis of the pathology and prompt interventions should be aimed at preventing or slowing down its progression. In this regard, the identification of suitable, least-invasive biomarkers (e.g., through blood tests) should be considered a priority. Moreover, it is more and more evident that prevention through specific diets and physical exercise will represent the key in the future to counteract cluster conditions that put the individual at greater risk for cognitive and physical decline, including, e.g., MetS [39].

**Figure 3.** During middle-age, women experience menopause that is characterized by the physiological drop in the protective effects of estrogen hormones. Such a drop leads to increased inflammation and adiposity, two conditions that might reinforce one another in either decreasing brain plasticity or increasing amyloid deposition or both. Curcumin administration in middle-aged pre-menopausal women has the potential to break this vicious cycle overall boosting anti-inflammatory defenses, counteracting fat-mediated insulin resistance and preserving brain plasticity (left panel). So far, no specific sex-differences have been found with regard to curcumin dose–response efficacy. However, it is important to stress that identifying sex-specific critical time windows throughout life to start curcumin administration might be equally important and might improve the chance to protect the brain and to counteract cognitive decline. Such a time window in women might be the middle-age, right before the beginning of menopause, when the organism still retains a certain degree of metabolic flexibility. Thus, the earlier the intervention (also through specific diets and physical exercise) the greater the chance to prevent the decay in mental and physical health (**right panel**). Figure adapted with permission from [150], Copyright © 2015 Christensen and Pike, and from [136,159]: © 2007 Springer International Publishing and © 2020 Published by Elsevier Ltd on behalf of IBRO.

#### **8. Conclusions**

In this review, we have touched upon a number of critical issues that should be taken into account when designing preclinical and clinical studies aimed at assessing curcumin efficacy on cognitive functions. Among these, one important factor that clearly needs to be tackled in the future has to do with increasing its bioavailability as well as controlling the impact of nutritional status/diet and lifestyle on curcumin's effects. Diet is also likely to influence microbiota status, thus, controlling for nutritional status will be crucial for effective future studies.

Another important point relates to the need to assess the effects of this natural compound on both males and females, both in preclinical and clinical studies. As dementia and AD show a much greater prevalence in the female population, it is imperative to address this issue by targeting both sexes/genders [151].

Moreover, given the overlap between the mechanisms of action of many compounds (see, e.g., curcumin and resveratrol), it will be important to target multiple molecular pathways to maximize the effects. Traditional medicines mostly use mixtures of phytochemicals, rather than individual compounds, which suggests the need to examine, with rigorous clinical trials, the role of plant mixtures on brain health [9,160].

Ultimately, in order to assess the effects of natural compounds, such as curcuma, we need to refine our ability to measure health (and the lack of) in a life-long perspective and to characterize the conditions for the transition from health to disease [39,73,135].

**Author Contributions:** A.B., B.C. and F.C. conceived the review, retrieved the references and drafted the manuscript. R.V. and R.M. contributed on the issue of curcumin bioavailability, biotechnologies and inflammasome, commented on gender issues and critically read the whole manuscript.

**Funding:** This work was supported by the JPI HDHL-INTIMIC-085-GUTMOM project within the ERA-NET Co-fund HDHL-INTIMIC (INtesTInal MICrobiomics) programme and HDHL-INTIMIC Knowledge Platform on Food, Diet, Intestinal Microbiomics and Human Health; F.C. received funding from the Italian Ministry of Health (Ricerca Corrente).

**Acknowledgments:** We thank Chiara Musillo for help in retrieving and selecting bibliographic entries; Paola Rossolino for management of external funding.

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

#### **References**


## *Review* **Obesity-Associated Inflammation: Does Curcumin Exert a Beneficial Role?**

**Rosaria Varì, Beatrice Scazzocchio, Annalisa Silenzi, Claudio Giovannini and Roberta Masella \***

Center for Gender-Specific Medicine, Gender Specific Prevention and Health Unit, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy; rosaria.vari@iss.it (R.V.); beatrice.scazzocchio@iss.it (B.S.); annalisa.silenzi@iss.it (A.S.); claudio.giovannini@iss.it (C.G.)

**\*** Correspondence: roberta.masella@iss.it

**Abstract:** Curcumin is a lipophilic polyphenol, isolated from the plant turmeric of Curcuma longa. Curcuma longa has always been used in traditional medicine in Asian countries because it is believed to have numerous health benefits. Nowadays it is widely used as spice component and in emerging nutraceutical food worldwide. Numerous studies have shown that curcumin possesses, among others, potential anti-inflammatory properties. Obesity represents a main risk factor for several chronic diseases, including type 2 diabetes, cardiovascular disease, and some types of cancer. The establishment of a low-grade chronic inflammation, both systemically and locally in adipose tissue, occurring in obesity most likely represents a main factor in the pathogenesis of chronic diseases. The molecular mechanisms responsible for the onset of the obesity-associated inflammation are different from those involved in the classic inflammatory response caused by infections and involves different signaling pathways. The inflammatory process in obese people is triggered by an inadequate intake of nutrients that produces quantitative and qualitative alterations of adipose tissue lipid content, as well as of various molecules that act as endogenous ligands to activate immune cells. In particular, dysfunctional adipocytes secrete inflammatory cytokines and chemokines, the adipocytokines, able to recruit immune cells into adipose tissue, amplifying the inflammatory response also at systemic level. This review summarizes the most recent studies focused at elucidating the molecular targets of curcumin activity responsible for its anti-inflammatory properties in obesity-associated inflammation and related pathologies.

**Keywords:** curcumin; obesity; inflammation; adipose tissue

#### **1. Introduction**

Curcumin, the main natural polyphenol found in the rhizome of Curcuma longa (turmeric) [1], has been recognized for thousands of years because of its medicinal properties and potential health benefits [2]. It is used worldwide in different forms: as spice, antiseptic, anti-inflammatory, preservative or coloring agent, as well as supplement in capsules or powder form [3]. It has been reported the beneficial effect of curcumin in various diseases, including inflammatory and degenerative conditions, cancer, dyslipidemia, metabolic syndrome (MetS), and obesity [4–7]. Several studies have also shown that most of the benefits are due to its antioxidant and anti-inflammatory activities [5]. Overweight and obesity are a major public health problem all over the world [8]. Obesity is caused by the imbalance between energy intake and energy expenditure, culminating in the excess of fat accumulation in the adipose tissue (AT) [9]. It is associated with a chronic low-grade inflammation that might represent the main factor linking obesity and the development and progression of various diseases including type 2 diabetes (T2D), dyslipidemia, heart diseases, stroke, and cancer [10,11]. AT, indeed, is recognized as an endocrine organ that secretes a number of cytokines and chemokines with regulatory and immune functions [12]. Dysfunctions of the secretory activity of AT, thus, most likely play a pathogenic role in the occurrence of the obesity-related pathologies [13]. In consideration of this, the current

**Citation:** Varì, R.; Scazzocchio, B.; Silenzi, A.; Giovannini, C.; Masella, R. Obesity-Associated Inflammation: Does Curcumin Exert a Beneficial Role? *Nutrients* **2021**, *13*, 1021. https://doi.org/10.3390/nu13031021

Academic Editor: Giuseppe Grosso

Received: 25 February 2021 Accepted: 18 March 2021 Published: 22 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

review examines specifically the possible role of curcumin in counteracting the activation of inflammatory pathways in AT. To this purpose, we conducted a comprehensive literature search until December 2020 in PubMed, using "obesity", "inflammation", "adipose tissue", "adipocyte" as key words in combination with "curcumin" and "dietary curcumin".

#### **2. Obesity, AT Dysfunction and Inflammation**

Obesity is characterized by an excessive AT expansion due to hyperplasia (increase in number) and/or hypertrophy (increase in size) of adipocytes, the major cellular component of AT. Although the main function of adipocytes is the storage and release of lipids, they secrete also active molecules that are used for intracellular signaling and to communicate with every organ system in the body. The second largest AT cellular component beyond adipocytes are resident immune cells that, in turn, play important roles in the maintenance of AT homeostasis. The intensity and complexity of these signal networks are highly regulated, differ in each fat pad, and are dramatically affected by various disease states. In conclusion, AT is an active endocrine organ, secreting a variety of hormones and metabolites that regulate systemic metabolism. When the imbalance in the storage of lipids by fat cells is established, alterations in secretive function occur and systemic metabolic dysfunctions might happen, such as in T2D, cardiovascular and liver diseases, and cancers. Through these cellular derangement and metabolic dysfunction, an excessive caloric intake contributes to a chronic low-grade inflammation, also known as 'metainflammation'. In particular, visceral AT accumulated in the abdominal seat, shows a disrupted balance between secreted pro- and anti-inflammatory factors, with increased levels of pro-inflammatory adipocytokines, including leptin [14], and decrease of anti-inflammatory adipokines, such as adiponectin [15]. These events all together cause local alterations of the AT environment and alter the normal cross-talk with other organs, such as liver, muscle, brain, and pancreas [16,17], which leads, as a further result, to metabolic dysfunctions, such as hyperinsulinemia and insulin resistance (IR) [11]. Furthermore, the polarization profile of the resident immune cells depends on the health status of the adipocytes [18]. Changes in the adipocyte secretion profile, in fact, trigger the recruitment and activation of immune cells [19]. In particular, in obese subjects, AT macrophages shift from an anti-inflammatory profile (such as that found in normal weight people) towards a pro-inflammatory phenotype [20,21] producing themselves an alteration in the production/activation of key factors that exacerbate local and systemic inflammation [22], such as tumor necrosis factor (TNF)α, interleukin (IL)-6, IL-1β, toll-like receptor (TLR) 4, and nuclear factor (NF)-κB, that may amplify the inflammatory state and favor the onset of pathologies [23–27]. The pro-inflammatory profile in obese individuals is evidenced by elevated serum levels of TNFα and IL-6, simultaneously with adiponectin and anti-inflammatory cytokines decrease [28,29]. The expression of the proinflammatory cytokines is regulated by the activation of the transcription factor NF-κB. This factor is stored in the cytoplasm as inactive form bound to the inhibitor IκBα that is, in turn, regulated by the inhibitor of κB kinase (IKK) complex consisting of 2 subunits, IKKα and IKKβ. Different stimuli, including growth factors, cytokines and foreign pathogens or molecules, such as lipopolysaccharides (LPS) and free fatty acids (FFA) [30], activate the IKK kinase complex inducing proteasomal degradation of IκBα and leading to the translocation of NF-κB in the nucleus, where it induces the expression of genes of various inflammatory mediators. Obese people show an increased activation of NF-κB pathway, most likely responsible for the increased pro-inflammatory cytokine release [31]. Among the pro-inflammatory compounds, it should be mentioned leptin; it is primarily produced by AT, its level increases in obese people and participates in the control of body weight by regulating food intake and energy expenditure [32]. On the other hand, adiponectin, produced almost exclusively by AT, circulates in high concentration in plasma and has anti-inflammatory properties probably related to the inhibition of NF-κB activation and, consequently, to the reduced synthesis of pro-inflammatory cytokine [33,34]. The secretion of anti-inflammatory adipocytokines is inhibited in visceral AT from obese patients and

subjects with MetS leading to a significant reduction in their plasma levels [35,36]. Despite the intense experimental work carried out, the exact molecular mechanisms responsible for the chronic low-grade metabolic inflammation in obesity are not completely clarified yet. However, with the identification of the nod-like receptor pyrin domain-containing (NLRP)3 inflammasome in AT, a new hypothesis has been formulated suggesting that it might be relevant for regulating obesity-associated inflammation and insulin sensitivity [37]. The NLRP3 inflammasome is a cytosolic molecular complex whose expression in AT directly correlates with body weight and aging, while its inactivation significantly mitigates metabolic disorders [38,39]. A number of exogenous and endogenous signals might act as NLRP3 inflammasome activator in AT leading to the production of pro-inflammatory cytokines [40,41] a potential mechanism linking an elevated intake of saturated fatty acids (SFAs) to the progression of metabolic diseases. In line with this, increased gene expressions of NLRP3 and its key effectors IL-1β and IL-18 have been observed in visceral fat of metabolically unhealthy individuals compared to those from lean healthy control or metabolically healthy obese individuals [42]. Furthermore, these inflammatory effects were suppressed, after weight loss, in the subcutaneous fat of patients with obesity and T2D, with consequent improvement in insulin sensitivity [38]. Studies have hypothesized a causal nexus between systemic inflammation and an increased release of FFA from AT in obese and insulin-resistant subjects [43,44]. Indeed, the direct drainage of free FA and adipokines from visceral AT to the liver can activate immune responses leading to the secretion of inflammatory compounds [17]. A potential mechanism of action through which FFA, and mainly dietary SFAs, can mediate AT dysfunctions contributing to the onset of inflammation involves the TLR4 [45,46]. TLR4 belongs to the TLR family and is expressed not only on leukocytes but also on many non-immune cells, including adipocytes, hepatocytes, and muscle cells. It has been hypothesized that FFA can bind and stimulate TLR4; thus, the elevated plasma level of FFA observed in obesity could activate TLR4. A recent research showed that the TLR4 activation can mediate inflammatory processes also through the impairment of adipogenesis which, in turn, elicit adipocyte and resident immune cell dysfunctions [47]. In conclusion, the onset of inflammatory processes linked to obesity and metabolic dysfunction in AT involves a number of different factors closely intertwined. The inflammation associated with obesity has been shown to derive from changes in the delicate crosstalk between adipocytes and macrophages due to an increased infiltration of macrophages into AT, the activation of a number of pro-inflammatory pathways, the alterations of adipokine production and increased expression and release of a panel of inflammatory cytokines. Understanding the molecular and metabolic switches that, starting from AT, lead to immune cells polarization towards inflammatory phenotypes may allow the definition of interventions capable of leading to the resolution of inflammation and blocking the sequence of events responsible for the occurrence of clinical complications in obesity. Targeting the key intracellular pathways underlying AT dysfunctions might represent a useful tool in counteracting obesity-related pathologies. From this point of view, the identification of potential protective activity of curcumin in positively modulating AT pro/anti-inflammatory balance has been gaining significant interest.

#### **3. Curcumin and Inflammation in Obesity**

Several studies carried out in humans have shown that curcumin attenuates inflammation in obesity and obesity-related diseases by rebalancing the equilibrium between antiand pro-inflammatory factors via different mechanisms due to the interactions of curcumin with a wide range of biomolecules, such as transcription factors, cellular receptors, growth factors, enzymes, cytokines, and chemokines [48,49]. Moreover, some reports have suggested that curcumin can enhance weight loss induced by diet and lifestyle intervention on overweight subjects with MetS ([50,51]. However, it should be considered that a main problem in the use of curcumin is its poor bioavailability. To increase curcumin bioavailability, different delivery systems including micelles, liposomes, phospholipid complexes, nanostructured lipid carriers, and biopolymer nanoparticles have been developed, as well

as the addition of piperine, a bioactive alkaloid extracted from the Piper species, which has been shown to effectively enhance the bioavailability of several nutritional supplements including curcumin [52].

#### **4. Curcumin Decreases Circulating Inflammatory Markers in Overweight/Obese Subjects**

There is an increasing evidence that curcumin treatment could be able to alleviate the altered pro-inflammatory mediator secretions present in obesity and related pathologies. In this section, data from human studies carried out on overweight and obese subjects with curcumin supplementation are collected and summarized. A research performed on 84 overweight or obese patients with non-alcoholic fatty liver disease (NAFLD) demonstrated that, curcumin supplementation with two 40 mg capsules/day after meals for 3 months, induces a decrease in many serum inflammatory markers, such as TNFα, high-sensitive C-reactive protein (hs-CRP), and IL-6 [53]. The same conclusions were reached by other studies carried out in obese/overweight people; specifically, curcumin administration (1 g/day) for 8 weeks reduces serum concentrations of TNFα, IL-6, and monocyte chemoattractant protein 1 (MCP-1) in males and females with diagnosis of MetS with respect to the placebo group [7]. In a randomized placebo-controlled clinical trial carried out on 60 adolescent girls undergoing to a slight weight-loss diet for 10 weeks, curcumin consumption (500 mg/day) was able to induce a significant decrease in hs-CRP and IL-6 compared to placebo supplementation [54]. In addition, it has been demonstrated that curcumin modulates circulating levels of IL-1β in thirty subjects randomized to receive curcumin (1 g/day) or a matched placebo for 4 weeks. Serum IL-1β was found to be significantly reduced by curcumin treatment. In contrast, no significant difference was observed in the concentrations of IL-6, and MCP-1 [55]. Finally, curcuminoids supplementation (300 mg/day) for 3 months in T2D patients led to a significant decrease in circulating FFA levels [56], that are considered a major factor linking obesity and inflammation [57–59].

#### **5. Curcumin Modulates Adipokines**

Adiponectin and leptin are two important adipokines released by adipocytes that have [18] several target organs including brain, liver, pancreas, muscle, immune system, and AT itself. They are involved in inflammation and immune response, showing, as stated above, adiponectin anti-inflammatory properties, leptin, on the contrary, pro-inflammatory ones [60]. Obese subjects are characterized by an imbalance of the two adipokines showing a low concentration of adiponectin and high levels of leptin in plasma [61]. Curcumin has been shown to increase the production of adiponectin [62]. To this regard, a systematic review [63] showed that curcuminoid administration significantly increased plasma adiponectin concentrations in randomized controlled trials. Specifically, in a double-blind randomized trial carried out over a 12-week period on 118 patients with T2D the effects of the daily administration of 1 g curcumin added with 10 mg piperine were compared to placebo. The treatment with curcumin plus piperine reduced serum levels of TNFα and increased serum level of adiponectin [64]. In another study, curcumin supplementation (1 g/day) for 6 weeks increased serum adiponectin concentrations compared to both curcumin-phospholipid complex (1 g/day) and placebo groups in 120 men and women with MetS [65]. In a randomized double-blind study 44 men and women with T2D were treated with curcumin 1500 mg/day or placebo for 10 weeks. At the end of the study, a significant increase in serum adiponectin concentration together with a decrease in the mean weight were observed in the curcumin group [66]. Conversely, no effect on adiponectin was seen in 22 young men randomly assigned to receive curcumin (500 mg/day) or placebo for 12 weeks. This finding might be determined by the low dose of curcumin used for the treatment [67]. However, the same amount of curcumin (500 mg/day) for 4 weeks reduced serum leptin and resistin and increased adiponectin content in 15 children and 15 adults [68,69]. Accordingly, elevated levels of adiponectin and decreased leptin levels were reported in diabetic men and women after 6-month intervention with a high dose of curcumin (1500 mg/day) [70]. Similar effects on serum levels of leptin were observed in

males and females with NAFLD treated for 12 weeks with even higher doses of curcumin (3000 mg/day) [71]. In conclusion, all the studies discussed show that curcumin supplementation contributes to rebalance pro- and anti-inflammatory factor production significantly increasing the levels of anti-inflammatory adipocytokines, such as adiponectin, and decreasing the pro-inflammatory ones, such as TNFα, IL-6, IL-1β, and MCP-1, counteracting the chronic inflammatory condition in overweight/obese subjects (Table 1).


**Table 1.** Effects of curcumin on inflammation in obesity: human studies.

**Study Design Subjects Treatment Duration Outcomes References** Randomized double-blind, placebo-controlled Obese (males, *n* = 22) 11 curcumin (500 mg/day) 11 placebo 12 weeks no change Adiponectin [67]

**Table 1.** *Cont.*

Abbreviations: ↑ Increases; ↓ Decreases; IL-6, interleukin-6; IL-1β, interleukin-1β; MCP-1, monocyte chemoattractant protein-1; TNFα, tumor necrosis factor α; FFA, free fatty acids; T2D, type 2 diabetes; MetS, metabolic syndrome; NAFLD; nonalcoholic fatty liver disease.

#### **6. Effects of Curcumin on Inflammatory Signaling Pathways**

Most of the potential molecular mechanisms responsible for the health effects of curcumin have been studied in animals and in vitro models using stabilized cell lines or human primary cells. Although the results obtained in this way cannot be completely extrapolated to humans, they allowed to suggest possible mechanisms of curcumin action that could explain the phenotypic effects evidenced by the studies carried out in humans (Figure 1). As regards animal studies, the anti-inflammatory effect of curcumin was first demonstrated in acute and chronic models of inflammation in rats and mice [72]. Specifically, in obese mice, curcumin treatment (3% by weight for 6 weeks) decreases NF-κB activity in liver tissue, associated with decreased hepatic expression of inflammatory molecules, such as TNFα and MCP-1. Curcumin-treated obese mice also show a decreased macrophage infiltration and an increased expression of forkhead transcription factor (Foxo)1 and adiponectin into AT, and higher circulating adiponectin levels [72]. In line with these results, an in vivo study performed in male rats, T2D insulin resistant because of high-fat diet (HFD) consumption, demonstrated oral administration of curcumin (80 mg/kg body weight) was able to improve insulin sensitivity by attenuating TNFα serum levels [73]. Moreover, dietary curcumin (4 g/kg diet added 2 days/week) attenuated the inflammatory response induced by HFD in mice through inhibiting NF-κB expression and JNK signaling pathway in epididymal AT [74]. Interestingly, administration of 0.1% curcumin associated with white pepper (0.01%) (Curcuma-P®) significantly down-regulated the proinflammatory cytokines IL-6 and TNFα, but did not modify IL-1β and MCP-1, in the subcutaneous AT of mice after 4 weeks of HFD. This effect was relatively tissue-specific and independent on macrophage infiltration. Indeed, the inflammatory cell infiltration in AT was not modified by Curcuma-P® supplementation [75]. Another interesting activity of curcumin has been demonstrated on endoplasmic reticulum (ER). The involvement of ER stress (with accumulation of misfolded proteins) in the release of FFA has been observed in several studies [76–78]. Curcumin has been demonstrated to mitigate ER stress in mice fed HFD and in primary adipocytes. Specifically, short-term HFD feeding (10 days) increased ER stress in mouse AT by increasing the expression of phospho-inositol-requiring kinase 1(p-IRE1) and phospho-eukaryotic Initiation Factor 2 (p-eIF2), two important indicators of ER stress. Oral administration of curcumin (50 mg/kg) counteracted the activation of IRE1 and eIF2 by reducing the phosphorylation, and consequently inhibiting the ER stress in vivo. Similarly, curcumin (0.1, 1, 10 μM) treatment inhibited IRE1 and eIF2 activation in mouse AT treated with 100 μM of palmitate (inductor of ER stress). Furthermore, curcumin administration reduced glycerol and FFA released from AT of HFD-fed mice blocking cAMP/PKA signaling via regulation of AMP-activated protein kinase (AMPK) [79], as well as significantly decreased plasma FFA levels in HF-induced obese rats [80]. Several in vitro models have been used to collect more detailed information about the potential molecular mechanisms of action through which curcumin exerts its effects. Curcumin has been shown to inhibit the activation of the pro-inflammatory NF-κB signaling pathway in several cell types, including human adipocytes and macrophages [48]. In adipocytes treated with TNFα to induce inflammatory processes, the contemporary treatment with 20 μM curcumin suppressed the degradation of IκBα, the NF-κB inhibitor, reducing, consequently, NF-κB translocation to the nucleus and significantly inhibiting the expression of TNFα, IL-1β, IL-6 and COX2 genes and IL-6 secretion [81]. In the same type of cells, curcumin also exerts a protective effect on hypoxia in a dose-dependent manner (5, 10, and 20 μM) reducing the secretion of

the inflammatory cytokines and protecting mitochondrial functions [82]. Besides a direct effect on adipocytes, curcumin has been shown to exert anti-inflammatory effects by counteracting the increased recruitment of macrophages in AT from obese mice [72,83]. Several studies have evidenced that curcumin treatment reduces macrophage invasion of AT in mouse models of obesity [62,72]. It has been shown that the cross-talk between adipocytes and macrophages in AT triggers and increases inflammatory responses in obesity including the increased production of MCP-1 and other inflammatory cytokines [84,85]. Curcumin treatment (0.1–10 μM) of Raw 264.7 macrophages incubated with the culture medium of mesenteric AT taken from obese mice, potentially able to induce an inflammatory response, significantly inhibited the production of TNFα, MCP-1, and nitric oxide, as well as the migration capacity of the macrophages with respect to the cells not treated with curcumin. Furthermore, 10 μM curcumin treatment significantly inhibited MCP-1 release from 3T3-L1 adipocytes [86]. Studies carried out in different cell systems strongly suggest that the anti-inflammatory activity of curcumin occurs by modulating NLRP3 inflammasome. In THP-1 macrophages treated with phorbol 12-myristate 13-acetate (PMA), an activator of NLRP3 inflammasome, curcumin (6.25, 12.5, and 25 μM) reduced NLRP3 inflammasome level, the activation of caspase-1 and the secretion of IL-1β in a dose-dependent manner, most likely down-regulating TLR4/NF-κB signal transduction pathway that is involved in NLRP3 inflammasome activation [87]. In mouse bone marrow-derived macrophages (BMDM) treated with nigericin, another NLRP3 inflammasome activator, the pre-treatment with curcumin (30–50 μM) inhibited caspase-1 cleavage and IL-1β secretion. The same results were observed in human macrophages. Specifically, differentiated THP-1 cells pretreated with curcumin showed reduced caspase-1 activation and IL-1β secretion after treatment with LPS and nigericin. The inhibition of NLRP3 activation by curcumin appears to be due to the suppression of K+ efflux [88].

**Figure 1.** Potential anti-inflammatory mechanisms of curcumin in obesity. VAT: visceral adipose tissue; FFA: free fatty acids; TNFα: tumor necrosis factor α; IL-6: interleukin 6; IL-1β: interleukin β; ER: endoplasmic reticulum; NF-κB: Nuclear transcriptor factor kappa B; MCP-1: Monocyte chemoattractant protein-1; NLRP-3: nod-like receptor pyrin domain-containing 3.

In conclusion, a growing body of experimental data supports the hypothesis that the beneficial effects of curcumin on obesity-related pathologies may be related to the suppression of IL-6, TNFα, IL-1β, and MCP-1 expression from adipocytes, the inhibition of macrophage recruitment in AT, and the inhibition of the inflammatory activity of the NLRP3 inflammasome [89] (Table 2).


**Table 2.** Effects of curcumin on inflammation in obesity: in vivo and in vitro studies.


**Table 2.** *Cont.*

Abbreviations: ↑ Increases; ↓ Decreases; IL-6, interleukin-6; IL-1β, interleukin-1β; MCP-1, monocyte chemoattractant protein-1; TNFα, tumor necrosis factor α; FFA, free fatty acids; HFD, high-fat diet; ER, endoplasmic reticulum; LPS, lipopolysaccharides; PMA, phorbol 12-myristate 13-acetate; Foxo1, forkhead transcription factor 1; NF-κB, nuclear transcriptor factor kappa B; JNK, jun N-terminal kinase; TLR4, toll-like receptor 4; NLRP3, nod-like receptor pyrin domain-containing 3.

#### **7. Conclusions**

The inflammation present in AT is involved in the development of various obesityrelated pathologies. The studies reported in this review clearly show that curcumin supplementation significantly decreases inflammatory cytokine production and increases adiponectin level in plasma of obese and overweight subjects. Furthermore, curcumin can regulate several molecular targets including transcription factors (NF-kB, NLP3), signaling pathways and other complex regulatory systems in AT resulting in the suppression/attenuation of the chronic low-grade inflammation. However, since curcumin is widely used as a supplement around the world because of its health promoting properties, further studies, both in vitro to better define the mechanisms of action, and in humans by controlled gender-based clinical trials to evaluate the real effectiveness, are mandatory. It should be reached, thus, that ultimate evidence on curcumin effects and highlighted possible differences in the response to curcumin treatment between women and men, allowing the definition of personalized advice about curcumin consumption.

**Author Contributions:** R.V., B.S., and R.M. provided substantial contributions to the conception of the work, as well as manuscript writing; C.G., A.S. contributed to the final discussion and revision of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This review received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing is not applicable to this article.

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

#### **References**

1. Jin, T.R. Curcumin and dietary polyphenol research: Beyond drug discovery. *Acta Pharmacol. Sin.* **2018**, *39*, 779–786. [CrossRef]

2. Kocaadam, B.; Sanlier, N. Curcumin, an active component of turmeric (Curcuma longa), and its effects on health. *Crit. Rev. Food Sci. Nutr.* **2017**, *57*, 2889–2895. [CrossRef] [PubMed]


## *Review* **Anticancer Mechanism of Curcumin on Human Glioblastoma**

**Shu Chyi Wong 1, Muhamad Noor Alfarizal Kamarudin <sup>2</sup> and Rakesh Naidu 1,\***


**Abstract:** Glioblastoma (GBM) is the most malignant brain tumor and accounts for most adult brain tumors. Current available treatment options for GBM are multimodal, which include surgical resection, radiation, and chemotherapy. Despite the significant advances in diagnostic and therapeutic approaches, GBM remains largely resistant to treatment, with a poor median survival rate between 12 and 18 months. With increasing drug resistance, the introduction of phytochemicals into current GBM treatment has become a potential strategy to combat GBM. Phytochemicals possess multifarious bioactivities with multitarget sites and comparatively marginal toxicity. Among them, curcumin is the most studied compound described as a potential anticancer agent due to its multi-targeted signaling/molecular pathways properties. Curcumin possesses the ability to modulate the core pathways involved in GBM cell proliferation, apoptosis, cell cycle arrest, autophagy, paraptosis, oxidative stress, and tumor cell motility. This review discusses curcumin's anticancer mechanism through modulation of Rb, p53, MAPK, P13K/Akt, JAK/STAT, Shh, and NF-κB pathways, which are commonly involved and dysregulated in preclinical and clinical GBM models. In addition, limitation issues such as bioavailability, pharmacokinetics perspectives strategies, and clinical trials were discussed.

**Keywords:** curcumin; glioblastoma; anticancer; molecular signaling mechanism

#### **1. Introduction**

Brain tumors can be classified into grade I and II (benign, low-grade), grade III (malignant, high-grade) such as anaplastic astrocytoma, and grade IV (highly aggressive and malignant) such as glioblastoma (GBM) [1]. GBM is the most common and aggressive form of malignant primary adult brain tumor [2]. In the United States alone, the annual age-adjusted incidence of GBM is 3.22 per 100,000 persons based on registry data from 2012 to 2016 [2]. Based on the 2016 WHO classification of the central nervous system tumors, GBM is classified as a grade IV diffuse glioma. GBM is further classified into isocitrate dehydrogenase-wildtype (IDH-wildtype), IDH-mutant, and not otherwise specified (NOS) [3]. IDH-wildtype or primary (de novo) GBM accounts for 90% of the total proportion of GBM cases [3]. The IDH-mutant or secondary GBM, which may arise from a lower grade diffuse glioma, only accounts for about 10% of the total GBM cases [3]. Primary GBM is more common in elderly patients (median age of 62 years), while secondary GBM preferentially arises in younger patients (median age of 44 years) [3].

The standard care for newly diagnosed GBM patients is surgical resection, followed by radiotherapy (60Gy in 30 fractions) with concurrent oral administration of temozolomide (TMZ), followed by six cycles of adjuvant [4]. Additionally, monoclonal antibody bevacizumab and other alkylating agents such as carmustine, lomustine, nimustine, and fotemustine are used in GBM treatment [4]. Unfortunately, these treatments often prove ineffective, given the poor prognosis outcomes of GBM with a five-year survival rate under 10% and a median survival rate of around 12 to 18 months [2,5]. The high infiltration

**Citation:** Wong, S.C.; Kamarudin, M.N.A.; Naidu, R. Anticancer Mechanism of Curcumin on Human Glioblastoma. *Nutrients* **2021**, *13*, 950. https://doi.org/10.3390/nu13030950

Academic Editors: Roberta Masella and Francesca Cirulli

Received: 14 February 2021 Accepted: 10 March 2021 Published: 16 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

degree of GBM often causes surgical resection incapable of fully resecting the GBM tumor, leaving the residual presence of microscopic foci [6,7]. Moreover, the GBM tumors often develop chemo- and radio-resistance with the formation of glioma stem cells, leading to GBM recurrence [8]. In TMZ-resistant GBM tumors, numerous molecular pathways such as nuclear factor kappa light chain enhancer of activated B cells (NF-κB), p53, and JAK-STAT are found to be commonly dysregulated [8]. In addition, several clinical complications such as pancytopenia, pyrexia, wound healing complications, multi-organ failure, or even death are observed following the chemo-radiation and immunotherapy treatment [8–10].

Thus, in recent years, scientists have been focusing on phytochemicals as potential therapeutic agents in cancer management to minimize drug toxicity and side effects. Flavonoids represent the most common and widely distributed phytochemicals in fruits and vegetables. Various flavonoids such as tannins, quinones, stilbenes, and curcuminoids possess antioxidant, anti-inflammatory, antiviral, antimutagenic, and, most importantly, anticancer properties [11,12]. Among them, curcuminoids (especially curcumin) have been gaining immense attention because of its anticarcinogenic, antitumor, antioxidant, and antiinflammatory actions [13–15]. Curcuminoids are a family of active compounds found in the turmeric rhizome (*Curcuma longa*), an Indian spice commonly used in cooking. Natural curcuminoids are composed of curcumin, bisdemethoxycurcumin, and desmethoxycurcumin in a proportion of 77:3:17 [16]. Among them, curcumin is the most abundant compound and has been widely studied as a potential therapeutic agent in chronic diseases, such as neurodegenerative, cardiovascular, pulmonary, metabolic, and autoimmune diseases [17]. For instance, curcumin was able to restore oxidative stress and DNA methyltransferase (DNMT) functions against diabetic retinopathy [15]. Curcumin also acts as a wound healing promoting agent by facilitating collagen synthesis and fibroblast migration [18]. Several pre-clinical and clinical studies also reported its anticancer effects in colorectal cancer [19,20], pancreatic cancer [21], lung cancer [22], and GBM [23]. Curcumin can modulate multiple cellular signaling pathways and molecular targets involved in GBM tumor growth, migration, invasion, cell death, and proliferation [24–27]. Retinoblastoma (Rb), p53, MAP kinase (MAPK), P13K/Akt, JAK/STAT, sonic hedgehog (Shh), and NF-κB pathways are the most common targeted dysregulated pathways found in GBM and modulated by curcumin [28–34]. Moreover, curcumin is highly lipophilic and able to cross the blood–brain barrier (BBB) [35,36].

To date, numerous review studies have suggested curcumin as a potential drug for GBM. However, a greater focus on curcumin's anticancer potential in molecular signaling pathways that are commonly dysregulated in GBM is needed to provide a more comprehensive understanding of its therapeutic effects. This review includes the initial until recent pre-clinical and clinical studies of curcumin's mechanisms of action in modulating several molecular pathways such as Rb, p53, MAPK, P13K/Akt, JAK/STAT, Shh, and NF-κB pathways. This review paper also discusses curcumin's related issues such as low bioavailability, pharmacokinetics, and the perspective strategies to overcome these issues.

#### **2. Dysregulated Signaling Pathways Associated with GBM Pathogenesis**

Almost all GBMs are found to have dysregulated Rb, p53, JAK/STAT, MAPK, P13K/Akt, Shh, and NF-κB pathways. Thus, the following section discusses the mechanisms of action modulated by curcumin via these molecular signaling pathways involved in GBM cell proliferation, apoptosis, cell cycle arrest, autophagy, paraptosis, oxidative stress, and cell motility. The reported observation of in vitro and in vivo studies of curcumin against GBM are summarized in Table 1.


**Table 1.** Signaling pathways and mechanism of actions targeted by curcumin in vitro and in vivo against glioblastoma (GBM).


#### **Table 1.** *Cont.*

#### *2.1. Retinoblastoma (RB) Pathway*

The RB pathway plays a central role in cell proliferation by regulating the cell cycle [56]. This pathway mainly consists of five components, which are CDKN2A/p16Ink4a, cyclin D1, cyclin-dependent protein kinases (cdk4/6), RB-family of pocket proteins (RB, p107, p103), and E2F [57,58]. CDKN2A/p16 is a negative regulator that competes with cyclin D1 to bind to and inhibit the activity of CDK4/6. This, in turn, can induce cell cycle arrest at the G1/S phase by inhibiting phosphorylation of RB protein by the cyclin D1/cdk4/6 complex. The unphosphorylated RB protein binds to E2F protein to inhibit the activation of E2F-regulated gene expression, thereby inhibiting cell cycle progression, DNA replication, and nucleotide biosynthesis [57].

Many of the important components of this pathway are frequently altered in many cancer cells, including GBM [59]. According to The Cancer Genome Atlas (TCGA) pilot project, most of the GBM acquired mutations include homozygous deletion or mutation of *CDKN2A/p16* and *RB1*, and amplification of *CDK4*, *CDK6*, and *cyclin D*, which are associated with the RB signaling pathway [60]. *RB* promoter methylation and gene silencing are found in GBMs and are more frequently reported in secondary GBMs than primary GBMs [61]. The inhibition of the RB pathway via silencing/suppression of its component proteins increases etoposide-induced DNA double strand breaks, p53 activation, and TMZ-induced GBM apoptosis [62–65]. Additionally, the inhibition of cyclin D1 can downregulate Pglycoprotein (pgp) expression, which may help to overcome chemoresistance in GBMs [63]. Thus, the RB signaling pathway is an important drug targeted pathway to improve GBM prognosis and patient outcomes.

As shown in Figure 1a, curcumin inhibits the RB signaling pathway by increasing the negative regulator CDKN2A/p16Ink4a activity, which then suppresses the phosphorylation of RB protein. To date, Chin-Cheng Su and colleagues demonstrated that curcumin significantly inhibited the RB pathway in DBTRG glial cells in a time- and concentrationdependent manner [28]. In this study, curcumin treatment upregulated CDKN2A/p16 and downregulated the phosphorylated RB protein. It has been shown that CDKN2A/p16 protein compete with cyclin D1 to bind to CDK4/6 protein, which then inhibits phosphorylation of RB protein. Unphosphorylated RB protein could not dissociate from its repressor E2F to permit transcription of G1 genes for proceeding from G1 to S phase.

#### *2.2. P53 Pathway*

P53 is a tumor suppressor protein that can activate cell cycle arrest or induce cell apoptosis to prevent damaged cells from further dividing and growing [66]. Following DNA damage, p53 is activated to induce transcription of p21Waf/Cip1, a cyclin-dependent kinase inhibitor [58]. This P21Waf/Cip1 protein can induce G1/S and G2/M arrest by binding to and inhibiting the activity of Cdc2, cyclin-CDK2, -CDK1, and CDK4/6 complexes. This allows the damaged cells to undergo DNA repair prior to mitosis. The p53 activity can be inhibited by MDM2, the transcription of which is induced by TP53 through a negative feedback loop [67]. However, P14ARF, which is located in part of the CDKN2A locus, can bind to and inhibit MDM2 from binding to the N-terminal transactivation domain of TP53 [58,68]. On the other hand, activated p53 protein can activate the pro-apoptotic BH3-only members of the Bcl-2 protein family [69]. These pro-apoptotic proteins bind and inhibit the pro-survival Bcl-2 proteins to initiate the pro-apoptotic multi-BH domain members of the Bcl-2 family, such as BAX and BAK, to induce cell apoptosis.

Dysregulation of p53 pathway in GBM is mostly due to *TP53* mutation, amplification of *MDM2*, or loss of expression of CDKN2A-p14ARF [70]. The TCGA project demonstrated that the p53 signaling pathway is altered in most GBM samples with the association of *TP53* mutation or homozygous deletion, *P14ARF* deletions, and amplification of *MDM2* and *MDM4* [60]. According to WHO, the *TP53* mutation is more commonly seen in secondary GBM and is higher in proportion to primary GBM [3]. The clinical study showed that most of the sample cells (from GBM patients with age around 56) were Bcl-2 positive, and most of the Bcl-2 positive cloned cells acquired chemoresistance [71]. Thus, various strategies have been developed to target the p53 pathway, such as inhibition of pro-survival genes or MDM2/p53 interaction, degradation of mutant p53, and restoration of wildtype p53 [72–74].

As shown in Figure 1b, curcumin upregulates the expression of p53, p21, and ING4 and increases the BAX:BCL2 ratio to induce cell cycle arrest and apoptosis. Curcumin upregulates the expression of p53 and p21 in a time- and concentration-dependent manner, which then induces G2/M arrest in DBTRG cells [28]. Curcumin induces p53 activity by upregulating p21Waf/Cip1 and ING4 protein expression [37]. The upregulation of ING4 expression can increase p53 acetylation at Lys-382 and protein stability [75,76]. The acetylation of p53 inhibits its interaction with MDM2, which eventually induces cell cycle arrest and apoptosis, as observed in U251 cells [37]. In a different study, curcumin enhances paclitaxel (PTX) activity in C6 cells by increasing TP53 and p21 gene expression. In response to the increase of TP53 and p21 gene expression, the cell population in the G0/G1 phase increases while the cell population in the S phase decreases, indicating G0/G1 phase arrest [38]. Moreover, curcumin suppresses A172 cell viability by inducing paraptosis through the regulation of genes associated with the endoplasmic reticulum (ER) stress response [40]. Interestingly, in this study, interaction network analysis (with IPA software) revealed that the altered levels of ER-related miRNAs interact with p53- BCL2 pathways. Thus, it was suggested that the p53-BCL2 pathway might be involved in curcumin anticancer mechanisms. Additionally, curcumin potentiates the cytotoxic and apoptosis-inducing effect of etoposide and TMZ through downregulation of p53 mRNAs and upregulation of BAX-Bcl-2 in T98G and U87MG cells [39,42,55].

However, a contradictory finding showed that curcumin did not induce cell cycle arrest, as it enhanced cyclin B1 and decreased p21 expression in the radioresistant KNS60 and U251MG(KO) cells [41]. These radioresistant cells usually have a high basal p53 level, but the expression of p53 decreased following curcumin treatment. These results showed that mechanism actions of curcumin in radioresistant GBM cells are different.

#### *2.3. JAK/STAT Pathway*

Cytokines and growth factors can activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway to regulate cell proliferation, differentiation, migration, and apoptosis. This pathway involves the activation of growth factor receptor kinases, phosphorylation, dimerization, and translocation of STAT proteins into the nucleus to activate the downstream target genes. JAKs (Jak1, JAK2, JAK3, Tyk2) are the cytoplasmic tyrosine kinases that relay intracellular signals originating from extracellular receptors [77–79]. Following JAKs activation and phosphorylation of the tyrosine residues on receptors, STAT is activated through its recruitment and binding to the phosphorylated tyrosine residues. The activation of STATs only lasts from a few minutes to several hours under normal physiological conditions. However, aberrant activation of STAT signaling is found in many GBM tissues compared with normal human astrocytes, white matter, and normal adjacent tissue to the tumor [29,80,81]. Studies showed that the inhibition of either JAK or STAT phosphorylation is associated with reduced levels of anti-apoptotic proteins, resulting in apoptosis in GBM cells [29,43,81–83].

As shown in Figure 1c, curcumin can inhibit STAT activation and its downstream target genes involved in cell proliferation, migration, and invasion. Curcumin inhibits JAK1,2/STAT3 tyrosine-phosphorylation, and STAT3 target genes such as *c-Myc*, *MMP-9*, *Snail*, *Twist*, and *Ki67*, which in turn decrease GBM cell migration, invasion, and proliferation [29]. In the same study, curcumin significantly decreased the tumor cell proliferation and growth of the mid-line crossing in the intracranially implanted tumor-bearing mice compared with the control diet. Moreover, curcumin treatment resulted in 15% and 38% tumor-free long-term survival in Tu-2449-bearing mice and Tu-9648-bearing mice, respectively, where all control mice died. Consistently, another study supported that curcumin is capable of inhibiting cell proliferation through the inhibition of STAT3 protein along with reduction *c-Myc* and *Ki-67* transcription in several glioma cell lines [43]. Moreover, curcumin can inhibit the DNMTanalogue M.Sssl to demethylate the *RANK* CpG sites and transcriptionally upregulate *RANK* gene expression in U251 cells [44]. This study further reported that curcumin induces *RANK* expression through STAT3 suppression. Activation

of RANK has been known to be associated with pro-apoptotic and anti-tumorigenesis activities [84,85]. Additionally, inhibition of STAT3 can result in suppression of STAT3-DNMT1 interaction, which then demethylates tumor suppressor gene promoters [86]. Other than that, curcumin decreases the Tyr705 and increases the Ser737 phosphorylated form of STAT3 in human patient-derived GSC lines [30]. This causes the inactivation of STAT3 proteins via suppression of its nucleus translocation, which suppresses the activation of its downstream target genes, such as *survivin*, which inhibits GBM cell proliferation.

#### *2.4. MAP Kinase Pathway*

The mitogen-activated protein kinase (MAPK) pathway is a three-layer signaling cascade. This MAPK cascade is comprised of MAPK3, which activates MAPKK2 through serine/threonine phosphorylation, which then activates MAPK through tyrosine/threonine phosphorylation within a conserved Thr–Xxx–Tyr motif in the activation loop of the kinase domain [87]. There are at least 11 members of the MAPK superfamily, which are divided into three main groups: the extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK), and p38s [88]. Generally, *ERK* genes are activated by growth factors and mitogen, and the signaling cascades include RAF as MAPKKK and MEK as MAPK. The activation of ERK signaling promotes cell growth, apoptosis, differentiation, and development [89]. While p38s and JNK are activated by stress, inflammatory cytokines, and growth factors [90], their signaling cascades include MEKK as MAPKKK and MKK as MAPKK. Activation of either *p38s* or *JNK* genes may support inflammation, cell apoptosis, cell motility, growth, and chromatin remodeling. ERK, p38, and JNK signaling pathways favor both anti-apoptotic and pro-apoptotic proteins, depending on the cell type and condition [91]. Hence, the aberrant activation or deactivation of this MAPK pathway can promote abnormal cell proliferation, contributing to tumorigenesis.

Studies showed that targeting the MEK-ERK1/2 pathway is one of the approaches to block adhesion of GBM cells onto gelatin/collagen component of ECM, therefore decreasing the proliferation and migration of GBM cells [92,93]. The high expression of p38 had a positive correlation with the glioma's malignancy grade, while suppressing p38 expression inhibited proliferation and induced apoptosis in GBM cells [94]. Ken-ichiro Matsuda and colleagues discovered that self-renewing stem-like GBM cells have elevated JNK phosphorylation levels, accompanied by increased c-JUN phosphorylation at the cognate JNK phosphorylation site [95]. Treatment with JNK inhibitor reduced the self-renewing ability of the stem-like GBM cells, suggesting JNK is needed for self-renewal in vitro and in vivo. Several studies showed that the inhibition of ERK, JNK, and p38 MAPK pathways induced GBM cell cycle arrest and inhibited cell proliferation [96–99].

Curcumin can modulate the MAPK signaling pathway to regulate cell proliferation, tumorigenesis, apoptosis, and inflammation, as shown in Figure 1d. So-Young Kim and colleagues discovered that curcumin potently inhibited glioma invasion by inhibiting all the MAPK pathways (JNK, p38, ERK), which then suppressed phorbol myristate acetate (PMA)-induced mRNA expression of *MMP-1, -3, -9,* and *-14* in U87MG and U373MG cells [45,46]. Overexpression of the matrix metalloproteinases (MMPs) facilitates migration and invasion of malignant brain tumor cells to the surrounding brain tissues. These MMPs are upregulated in human malignant gliomas [100]. Among the MMPs, MMP-9 is the most common enzyme that promotes brain tumor invasion and is frequently found in GBM [101]. MMP-1 protein level is increased with the tumor grade and correlated with increased glioma invasiveness [102]. At the same time, the activation of MMP-3 can degrade the brain's hyaluronic acid-rich matrix, which leads to the invasion and migration of tumor cells [103]. Additionally, MMP-14 is a membrane-bound protease that can remodel the ECM to stimulate proMMP-2 activation. Curcumin treatment (10 μM) inhibits cell invasion by more than 90% in U87MG and U373MG cells [45].

In contrast, curcumin increases the phosphorylated ERK, p38, and c-Jun proteins levels, which decreases GBM stem cell (GSCs) proliferation, sphere-forming ability, and colonyforming potential [30]. Curcumin can also promote MAPK pathway activation through the

induction of reactive oxygen species (ROS) [30]. The production of intracellular ROS can induce the activation of ERK and p38 MAPK pathways through oxidative modification of intracellular kinases and inactivation of the MAPK phosphatases [104,105]. Moreover, curcumin induced Egr-1 expression through the activation of p38, ERK, and JNK pathways, which mediated the transactivation of Elk-1 in U87MG human GBM cells [47]. Egr-1 binds directly to the p21 promoter to stimulate p21 transcription, inhibiting CDK activity and resulting in cell cycle arrest. Elk-1 is the direct target gene of the p38, ERK, and JNK pathways, which can form a complex with serum response factor on the serum response element of the Egr-1 promoter to activate the p53-independent transcriptional activation of p21Waf/Cip1 protein. Additionally, curcumin can induce autophagy in GBM cells through the inhibition of the ERK1/2 pathway [48]. Another study suggests that the inhibition of the ERK pathway can lead to suppression of TORC1, which plays an important role in inhibiting autophagy initiation through the phosphorylation of Atg13, ULK, AMBRA, and Atg-14L [106]. In a C6 orthotropic xenograft, curcumin suppressed the phosphorylated JNK1 and JNK2 levels, which decreased lipopolysaccharide-induced CCL2 production [49]. The overexpression of CCL2 can contribute to GBM progression by inducing encephalopathy with mild perivascular leukocyte infiltration, impaired BBB function, and increased expression of proinflammatory cytokine expression [107].

#### *2.5. P13K/AKT Pathway*

The phosphatidylinositol-3-kinase (P13K)/Akt signaling pathway is critical in regulating cell growth, cell cycle arrest, apoptosis, and mRNA translation to maintain normal physiological conditions. There are three different classes of P13Ks, namely Class I, II, and III, which are categorized accordingly to their different structure and specific substrates [108,109]. Class I P13Ks are heterodimers consisting of a p110 catalytic subunit and p85 adaptor subunit [110,111]. Notably, Class 1 P13Ks is the most common type of P13K, which is incriminated in human cancer. The binding of cytokines or growth factors to the corresponding receptors results in the tyrosine residue autophosphorylation, followed by P13K binding protein recruitment. Upon allosteric activation of the p110 catalytic subunit, P13K catalyzes the phosphorylation of PtdIns(4,5) P2 (PIP2) to PtdIns(3,4,5) P3 (PIP3), which then recruits a subset of signaling proteins with pleckstrin homology (PH), such as AKT and PDK1, to initiate cell proliferation pathways. Protein-phosphatase and tensin homologue (PTEN) act to dephosphorylate PIP3 into PIP2 to prevent activation of the downstream kinases [110,111]. One of Akt's common target proteins is mTOR, which regulates cell growth and proliferation by promoting biosynthesis of multiple proteins such as cyclin D1, HIF, and VEGF [110]. The mTORC1 activates S6K and inactivates 4EBP1, promoting the production and translation of proteins to promote cell growth. While the mechanism of mTORC2 is less well clarified, it has the same responsibility in promoting cell proliferation [110].

Studies have reported that mutations of the core genes involved in P13K/AKT pathways are commonly found in human GBM tissues [60,112]. Alfeu Zanotto-Flho and colleagues reported that the P13K/Akt pathway are highly upregulated (seven- to eight-fold) in C6 and U138MG cell lines compared to the normal astrocytes cells [33]. Consistently, data from the TCGA pilot project showed that most of the GBM samples acquire homozygous deletion or mutation of *PTEN*, *P13K* mutation, and amplification of *AKT* and *FOXO* genes [60]. Another report indicated that genetic alterations such as loss of heterozygosity (LOH), mutation, and methylation have been identified in most GBM patients. LOH or *PTEN* mutation is positively associated with the poor survival of GBM patients [113]. It was reported that the delivery of Akt small-molecule inhibitor to inhibit the P13K/AKT pathway effectively suppressed the growth of both stem and non-stem GBM cell populations [114].

As seen in Figure 1e, the P13K/Akt signaling pathway and its key molecular targets are inhibited by curcumin to prevent GBM progression. Curcumin inhibits 80% of the P13K/Akt pathway's constitutive activation by suppressing the phosphorylation of Akt proteins on Ser473 [33]. Inhibition of the P13K/Akt pathway resulted in the induction of G2/M phase arrest as an early step of the apoptotic mechanism, which could probably explain how curcumin spared the non-transformed and its selectivity towards the tumor cells. In the same study, curcumin decreased GBM tumor size and increased apoptotic tumor cells in C6-implanted Wistar rats. Most importantly, curcumin did not cause any tissue toxicity in the rats' liver, kidney, lungs, or heart. Other than that, curcumin inhibited the P13K/Akt pathway by increasing PTEN expression, which decreased p-Akt and pmTOR expression, leading to cell apoptosis [31]. In the same study, curcumin also inhibited GBM tumor growth by increasing PTEN protein expression in the U87 xenograft model.

Additionally, curcumin induces autophagy by inhibiting the AKT/mTOR/p70S6K pathway in GBM cell lines and xenograft models [48,50]. In these studies, curcumin significantly decreased the levels of P13Kp85, phosphoP13Kp85, total Akt, p-AKT, mTOR, and p-mTOR. mTOR is not only a major effector of cell growth and proliferation, but it can also inhibit autophagy events in its active form [115]. Thus, inhibiting expression of P13K and AKT, which regulate mTOR expression, is a feasible strategy to induce autophagy– cell death in GBM cells. Notably, curcumin downregulates Bcl-2 and upregulates BAX, leading to the release of cytochrome-c and caspase-3 activation. Curcumin also enhances the anti-cancer effects of nimustine hydrochloride (ACNU) against GBM by inhibiting the phosphorylation of P13K and the AKT (serine/threonine) [51,116].

#### *2.6. Sonic Hedgehog (Shh) Pathway*

The hedgehog (Hh) signaling pathway is critical for embryonic development, organogenesis, regeneration, and homeostasis for adult tissue [117]. There are three main types of Hh proteins, which are sonic hedgehog (Shh), Indian hedgehog (Ihh), and desert hedgehog (Dhh). The activation of the Shh pathway can occur either through canonical or non-canonical signaling pathways [117]. The canonical Shh activation occurs by ligand-dependent interaction when Shh binds to the patched transmembrane receptor (PTCH) [117,118]. Following this binding, PTCH is incapable of inhibiting the second transmembrane protein, smoothened (Smo). Smo signals the suppressor of fused (SUFU), which is the negative regulator of glioma-associated oncogene homologue (GLI), to release and activate GLI. The activated GLI translocates into the nucleus and modulates downstream gene expression. On the other hand, the non-canonical Shh activation occurs through either GLI-independent mechanisms or Smo-independent mechanisms [117,118]. In the GLI-independent mechanism, Smo is no longer inhibited by the PTCH, and therefore it can stimulate the release of calcium ions from the ER to control the growth of the actin cytoskeleton [118]. In contrast, the Smo-independent mechanism is involved in cyclin B activation to increase cell proliferation and survival [118].

Under normal physiological conditions, the Shh pathway is minimally active in differentiated adult tissue, as it is a highly conserved development pathway. The Shh pathway is frequently associated with GBM tumorigenesis [119–122]. Studies have reported that most of the GBM patient tissues samples exhibited an aberrant activation of Hh signaling with the presence of GLI1 in both nucleus and cytoplasm [119,123]. Among GLI family members, overexpression of GLI1 is mostly associated with poor prognosis in several cancers, including GBMs [32,124–127]. GLI1 protein can upregulate several target genes such as *PTCH1, CycD1, MYC, Bcl-2, NANOG*, and *SOX2* to promote cell proliferation, apoptosis, angiogenesis, and stem cell self-renewal [128–130]. A study showed that inhibiting GLI1 alone significantly decreases the metabolic activity of GBM cells to reduce chemoresistance [119]. This study also revealed that inhibiting the expression of GLI1 proteins can elevate the nuclear p53 level in U87MG cells. Additionally, the overexpression of Smo is significantly associated with poor prognosis in GBM patients [131]. Smo expression inhibition, which suppresses GBM proliferation, migration, invasion, and tumorigenesis, further supports this observation [132].

As shown in Figure 1f, curcumin is a potent inhibitor of the SHH/GLI signaling pathway by downregulating the Shh, Smo, PTCH, and GLI protein levels and its downstream

target genes such as *cyclin D1, Bcl-2, and Foxm-1* [32,52,133]. Curcumin inhibits GBM cell proliferation, colony formation, migration, and induced apoptosis through downregulation of both mRNA and protein levels of SHH/GLI1 signaling (Shh, Smo, and GLI1) in U87 and T98G cells [32]. Curcumin also inhibits GLI nuclear translocation, which deactivates its downstream target genes including *cyclin D1, Bcl-1*, and *Foxm-1*. The combination treatment of curcumin and miR-326 can further reduce the tumor volume and prolong the survival period of U87-bearing mice by inhibiting GLI1 proteins compared with miR-326 or curcumin treatment alone [52]. In the same study, curcumin-treated GBM cells significantly decreased the expression of GLI1 protein, and this observation was enhanced with combination treatment with miR-326. The curcumin and miR-326 treatment also increased the expression of caspase-3 cleaved anti-poly ADP ribose polymerase 1 (PARP-1) caspase-in GBM cells. Simultaneously, the pro-survival proteins BCL-XL, MCL1, and RIP1 were decreased compared to the control and curcumin only-treated GBM cells.

#### *2.7. NF-κB Pathway*

NF-κB is a family of highly conserved transcription factors that regulate the transcription of various genes involved in cellular activities. There are four members under this NF-κB family: NF-κB1(p50/p105), NF-κB2(p52/p100), Rel-like domain-containing protein A (RelA/p65), and c-rel [134]. They form a dimeric complex (either homodimers or heterodimers) and bind to the specific sequences of DNA called response elements (RE) for the transcription of gene involved in cell proliferation, apoptosis, and inflammatory response [133,135]. NF-κB activation can occur through two major signaling pathways: the canonical and the non-canonical NF-κB signaling pathways [136,137]. The canonical pathway is mediated through the nuclear translocation of p50, RelA, and c-Rel into the nucleus and binding to the targeted DNA sequences. In contrast, the non-canonical NF-κB pathway selectively responds to stimulus and activates p100-sequestered NF-κB members, predominantly via translocation of NF-κB p52 and Rel B into the nucleus. NF-κB members normally bind to the DNA sequences of anti-apoptotic, pro-survival, and immune response genes. Several studies have demonstrated that human GBM cells have aberrant NF-κB activity to maintain their tumorigenic activity [138–143].

NF-κB p65 subunit is overexpressed in 81% of 69 samples of GBM and is frequently noted in high-grade compared to low-grade astrocytomas [139]. The constitutive activation of NF-κB p65 is detected in 93% of the GBM cells as compared to normal astrocytes. Studies conducted by Baisakhi et al. showed that inhibition of NF-κB activity resulted in decreased IL-8 transcription, which then inhibited GBM cell invasion and migration [140]. A study conducted by Denise Smith et al. demonstrated that GBM cells that are transfected with short hairpin inhibitory RNAs of RelA and c-Rel for six days displayed reduced tumor growth, signifying the role of RelA and c-Rel in GBM [144]. These studies highlight the importance of inhibiting the overactivation of NF-κB subunits as molecular targets in GBM.

Curcumin modulates the NF-κB pathway to confer the anti-inflammation, antiproliferation, and apoptotic activity in GBM cells, as shown in Figure 1e. Curcumin can downregulate NF-κB activity by decreasing the expression of anti-apoptotic protein BclxL in GBM cell lines [33]. Reducing the Bcl-xL triggers mitochondrial depolarization, which precedes the losses in mitochondrial membrane integrity. This suggests that curcumin induces mitochondrial-mediated apoptosis in GBM following the inhibition of NF-κB pathways. Other than mitochondrial depolarization, curcumin promotes cell cycle arrest in the G2/M phase prior to cell apoptosis. Most importantly, curcumin acts irrespective of the p53 or PTEN mutational status of the cells. Both PTEN and p53 mutated cells had the same experimental outcomes compared with the wild-type cells after being treated with curcumin. This shows that curcumin exerts p53-independent cell death via inhibition of NF-κB pathways. In the same study, the inhibition of NF-κB by curcumin increases the number of apoptotic cells in tumors, further reducing the tumor size and hemorrhagic areas in C6-implanted Wistar rats. This was in line with other studies showing that curcumin increases the IκB inhibitor proteins and decreases the expression of NF-κB-regulated

genes that contribute to GBM chemoresistance [51,53,145]. Curcumin also enhances the anticancer effect of nimustine hydrochloride (ACNU) by suppressing the phosphorylation of IκB, p65, and p50, which then decreases COX-2 expression [51]. Additionally, curcumin's antiproliferative activity might be facilitated through the downregulation of cyclin D1, since the promoter of cyclin D1 is regulated by NF-κB [54]. In the study conducted by Tzuu-Yuan and colleagues, curcumin increased NF-κB transcription factor inhibition in a concentration-dependent manner in GBM 8401 cells [55].

Curcumin improves the cytotoxic effect of PTX by reducing the phosphorylation of IκB and suppressing NF-κB p65 nuclear translocation to inhibit cell growth in C6 rat glioma cells [38]. Furthermore, curcumin upregulates the pro-apoptotic molecular Smac/Diablo to suppress NF-κB and IAPs (cIAP-1 and cIAP-2), which induces apoptosis [42]. Studies suggest a positive feedback system between NF-κB and IAPs, as IAPs can be upregulated by NF-κB and vice versa [146–148]. Hence, downregulation of both NF-κB and IAPs protein might further suppress GBM tumorigenesis.

#### **3. Issues of Curcumin Bioavailability and Methods to Overcome Them**

Despite the promising anticancer mechanisms, curcumin efficacy is hindered by its low bioavailability. Various studies have reported that very low curcumin concentration was detected in blood, tumors, or extraintestinal tissues [149–151], which may be due to the poor absorption, rapid metabolism, chemical instability, and rapid systemic elimination characteristics of curcumin [16]. A study reported that orally-administered curcumin at a dose of 500 mg/kg only had 0.06 μg/mL maximum serum concentration, indicating only 1% oral bioavailability [152]. Due to its chemical structure, curcumin has low solubility in neutral or acidic pH. It is fully protonated, unlike in alkaline conditions where it can be hydrolyzed, especially in the intestinal (pH 6.8). Additionally, rapid metabolism and systemic elimination happen through the formation of glucuronides and sulphates by conjugation in the intestine. Studies reported that free curcumin was undetectable, but curcumin glucuronides and sulphates were highly detected in most of the subjects' serum samples who had been administered with curcumin, and this indicates rapid metabolism of curcumin [153,154].

The first step of pharmaceutical strategies is to improve curcumin solubility and its absorption to overcome this problem. The incorporation of curcumin in solid dispersion, nanoparticles, micelles, conjugates, liposomes, and phytosomal formulations have increased curcumin's solubility and absorption rate in GBM cells [155–161]. Studies showed that curcumin-loaded noisome nanoparticles (CM-NP) can more effectively suppress the viability, proliferation, and migration of GSCs by inducing cell cycle arrest and apoptosis [155]. The CM-NP also efficiently increases ROS-suppression of tumor growth and inhibits monocyte chemoattractant protein 1 (MCP1) to reduce the invasiveness of GSCs compared to curcumin alone. Additionally, rats injected with curcumin-loaded PLGA nanoparticles have significantly smaller tumor size after five days of injection, while the group injected with curcumin alone displayed no significant change [156]. Another in vivo study reported that the combination of antisense-oligonucleotide against miR-21 with curcumin-loaded DP micelle complex reduced the tumor volume more effectively than single therapy curcumin or miR21ASO alone [157].

Additionally, relative to natural curcumin, solid lipid curcumin particles can promote cell death and DNA fragmentation by increasing the levels of caspase-3, Bax, and p53 with downregulation of Bcl-2, c-Myc, and Akt proteins in GBM cell lines [159]. The antibody-conjugated biodegradable polymeric nanoparticles (Mab-PLGA NPs) could enhance the photodynamic efficiency of curcumin on DKMG/EGFRvIII GBM cells compared to curcumin loaded biodegradable polymeric nanoparticles alone (56% vs. 24%) [158]. Furthermore, curcumin analogue induces FBXL2-mediated AR ubiquitination, ROS, lipid peroxidation, and suppression of glutathione peroxidase 4 to inhibit growth of TMZsensitive and -resistant GBM in vitro and in vivo [160].

Moreover, curcumin liposomes can significantly improve the anti-tumor effects of curcumin by enhancing the uptake effects, apoptosis effects, and endocytic effects of C6 glioma cells and C6 glioma stem cells. Curcumin liposomes were also shown to inhibit tumor growth and increase the survival period of brain glioma-bearing mice [161]. Other than that, a study showed that curcumin-loaded targeted liposomes cross the BBB two-fold higher than the non-targeted liposomes loaded with curcumin [162]. Curcumin-loaded targeted liposomes can more effectively inhibit GBM tumor growth and increase the survival rate of U87 GBM tumor-bearing mice compared to free curcumin as well as the non-targeted liposome-loaded curcumin [162]. Additionally, curcumin phytosome meriva (CCP) has been shown to improve curcumin bioavailability [163], which then help to activate natural killer cells and mediate elimination of GBM and GBM stem cells [164]. Based on the results of the preclinical studies, the use of the conjugate, nanoparticles, micelles, solid lipid, analogues, liposomes, or phytosomal formulation could certainly be clinically developed to benefit GBM patients.

#### **4. Clinical Trials**

Currently, there is only one clinical study investigating the curcumin effects on 13 newly diagnosed pre-operative GBM patients [23]. In 2014, this clinical study reported the highest serum and intratumoral concentrations of curcumin detected using the micellar curcumin formulation. It was reported that the intratumoral concentration of curcumin detected might not be sufficient to cause short-term antitumor effects. Still, it might help to control tumor growth in a long-term way. Moreover, intratumoral inorganic phosphate was significantly increased by curcumin. This might indicate increased demand for highenergy phosphates or mitochondrial dysfunction, since inorganic phosphate is used for ATP generation [23]. In addition, the side effects of taking curcumin are significantly less severe than the current chemotherapeutic drugs [23]. Thus, the oral administration of micellar curcumin is relatively safer and well-tolerated. However, this clinical trial only involved a small number of patients with a small dose of curcumin. Further clinical trials should be carried out to strengthen the statistical validity with a larger sample size. In the future, phase I/II clinical trials should be carried out to determine the safety and ideal dosage for GBM treatment, and phase III to IV to examine curcumin's efficacy and potential side effects with a larger sample population. Other than that, randomized controlled trials can be carried out to avoid bias and provide higher accuracy results.

#### **5. Conclusions and Future Perspectives**

Taken together, curcumin possesses the ability to modulate various core signaling pathways that are commonly dysregulated in GBM. However, among these signaling pathways, a greater emphasis on Rb and Shh pathways could be of focus for future pre-clinical studies, since the current data are still limited. Additionally, the contradictory findings on curcumin modulation of the p53 pathway warrant future investigation and suggest that curcumin use may be selective against radioresistant GBM tumor. Nevertheless, the combination of curcumin with standard chemotherapeutic drugs mainly results in the modulation of multiple signaling pathways that promote their anti-cancer effects. Curcumin's ability to modulate the major signaling pathways while promoting the efficacy of standard chemotherapeutic drugs warrants its use as a potential nutraceutical-based adjuvant drug for GBM treatment.

Since the clinical studies of curcumin in GBM patients are lacking, it is worthwhile for future clinical studies to incorporate curcumin as a potential neo-adjuvant in GBM. Although issues such as bioavailability, poor absorption, and rapid systemic elimination may hinder its efficacy, the pre-clinical use of nanodelivery has shown great promise while increasing the efficacy of curcumin and chemotherapeutic drugs. Considering this, a greater emphasis should also be given towards the nanoformulations of curcumin in future clinical studies, in combination with the standard chemotherapeutic drugs. Therefore, the multimodal modulation of signaling pathways via nanoformulation of targeted curcumin delivery that can synergize chemotherapeutic drugs efficacy may provide a clinical perspective in GBM therapy.

**Author Contributions:** S.C.W. designed the outline of the manuscript and wrote the manuscript. R.N. designed the outline of the manuscript and edited and revised the manuscript. M.N.A.K. designed the outline of the manuscript and edited and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a JCSMHS—BRIMS collaboration grant, Monash University Malaysia.

**Acknowledgments:** The authors are thankful to Monash University Malaysia, for providing financial support to conduct this study.

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

#### **References**

