1. Introduction

Quercetin (3,3’,4’,5,7-pentahydroxy flavone) is one of the main components of the polyphenol family of flavonoids ^[1] and it is mostly present in fruits, vegetables and some plant-derived beverages, such as wine or tea ^[2]. This flavonoid has many beneficial properties on human health ^[2], being associated its biological activity with the presence of five hydroxyl groups on the ring structure ^[1]. A number of studies have investigated quercetin effects on cellular processes involved in different human pathologies ^[3][4]. Anti-inflammatory, antioxidant and anticancer activities are some of the mainly described quercetin mechanisms of action ^[1][2][5]. Besides, therapeutic potential of this flavonoid has been evaluated in a broad variety of human disorders, including diabetes, cardiovascular ^[3], neurodegenerative ^[3][4][6] and Alzheimer’s diseases ^[6]; and positive actions on blood vessel pressure, intestinal microbiota and kidney disfunction ^[5], among others, were also related to quercetin efficacy.

Liver injury is largely caused by obesity or metabolic syndrome, in addition to high alcohol consumption ^[5][7]. Hepatocyte damage eventually contributes to the development of liver disorders including steatosis, alcoholic and non-alcoholic steatohepatitis which could cause non-alcoholic fatty liver disease (NAFLD), liver inflammation and hepatic fibrosis ^[5][7]. Hepatic chronic damage often leads to progression to liver cirrhosis and, in most cases, to hepatocarcinoma (HCC) ^[5][7]. In addition to the aforementioned beneficial effects, quercetin has been shown to exert multiple hepatoprotective actions through lipid biogenesis modulation, mitochondrial biogenesis activation ^[8] and the increase of cellular antioxidants and insulin sensitivity ^[5]. As part of its hepatoprotective ability, this flavonoid has demonstrated to reduce oxidative stress and inflammatory response in liver damage caused by alcohol and different toxic compounds (e.g. ethanol, metals and pesticides) ^[9]. Generation of an inflammatory and fibrotic microenvironment are key mechanisms produced in chronic-injured liver by hepatic stellate cells, and quercetin is able to abrogate its activation and modulate its polarization, restraining liver cells alteration ^[10]. Along with this, regulation of liver cell pathways involved in cell proliferation, differentiation and extracellular matrix synthesis is associated with quercetin-derived positive effects in the prevention of NAFLD ^[11][12] and liver fibrosis ^[7]. Some studies have also proved its beneficial activities against liver cirrhosis development and pulmonary associated complications ^[13][14], what makes quercetin a promising agent for the improvement of the outcomes in liver pathologies therapy ^[9].

HCC is the most common primary liver cancer and the sixth tumor with higher incidence, ranking as the fourth deadliest neoplasm worldwide ^[15]. Liver damage caused by different etiologic agents, mainly hepatitis C and B virus (HCV and HBV, respectively), contributes to HCC development through the stages of liver fibrosis and cirrhosis, which can take from years to decades ^[15]. Its complex pathogenesis and molecular heterogeneity hinder HCC early diagnosis making curative treatments not possible ^[15]. In these cases, systemic therapy is used, being available two tyrosine kinase inhibitors (TKIs), sorafenib and lenvatinib, in the first-line setting for advanced HCC ^[16]. Regardless its effectiveness, liver cancer cells are able to develop sorafenib resistance after sustained administration ^[17], where several TKIs (regorafenib and cabozantinib) and monoclonal antibodies (nivolumab, pembrolizumab and ramucirumab) have been recently approved ^[16]. Considering toxicity and adverse reactions caused by these chemotherapeutic agents, some investigations have focused on the study of antitumor effects of natural compounds against HCC, such as resveratrol, curcumin and melatonin ^[18][19][20].

2. Antitumor properties of quercetin as single agent against HCC

2.1. Antiproliferative activity of quercetin

Quercetin antitumor effects have been described in different cancer types, including HCC ^[1], being its antiproliferative effect widely observed in several researches ^{[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]}. It has been described that quercetin-derived inhibition of liver cancer cell growth could be mediated by the disruption of different pathways, including protein kinase B (Akt)/mammalian target of rapamycin (mTOR) ^[22][23][28], mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase 1/2 (ERK1/2) ^[26][32] and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling routes ^[21][34]. Another lesser known molecular pathways have also been altered by quercetin, including Src homology domain 2 containing tyrosine phosphatase-1/2 (SHP-1/2), transcription factor specificity protein 1 (Sp1), phosphatidyl inositol 3 kinase (PI3K) and protein kinase C (PKC) routes ^[28][30][34]. These findings evidence the high number of mechanisms of actions that are associated with the antiproliferative activity of quercetin.

2.2. Cell cycle modulation by quercetin

Otherwise, though cell cycle regulation is a common mechanism found to be altered in HCC cells, publications that have evaluated the effects of quercetin in this process show contradictory results. As common feature, quercetin was able to arrest cell cycle, either in G0/G1 or G2/M phases; however, modulation of cell cycle markers such as p53, p21, p27 and cyclin D1 by quercetin differs among the investigations performed ^{[21][24][27][30][35][36][37][38]}.

2.3. Apoptosis induction by quercetin

Apoptosis has been clearly established as one of the mechanisms of quercetin-induced cell death in HCC ^{[21][23][27][28][29][30][36][37][38][39][40]}, as it was demonstrated by the increase in proapoptotic proteins expression, such as Bax and cleaved caspases-3 and -9 ^{[21][23][26][28][29][30][37][38][39][40]}, and the opposite trend in the levels of antiapoptotic proteins, for instance Bcl-2 and Mcl-1 ^{[23][26][29][30][36][37][40]}. Autophagy is a self-recycle process by which damaged cell components are degraded, and it has been associated with either prosurvival or antitumor role depending on the tumor cell context ^[21][41]. In this case, two studies observed that quercetin treatment promoted autophagy in HCC cells, being related to apoptosis induction and suppression of tumor progression ^[21][23].

2.4. Antiangiogenic effects of quercetin

Angiogenesis and metastasis are tightly associated with HCC progression and represent very common targeting processes in cancer treatment ^[15]; however, there is a lack of studies evaluating quercetin effects on cell migration and invasion ability ^[21]. It was reported that this flavonoid regulated the expression of epithelial and mesenchymal markers in favor of first ones, abrogating epithelial-to-mesenchymal transition (EMT) and invasiveness of HCC cells ^[21]. Similarly, another study revealed a greater antiangiogenic activity by combination of quercetin and sorafenib in an in vivo HCC model ^[42], which suggest a promising role of quercetin not only as cytostatic agent but also in preventing recurrence-associated processes of angiogenesis and invasion.

2.5. Other antitumor actions of quercetin

In addition to the described properties, quercetin has shown to act as an antioxidant agent in human HCC models ^[24][28][43], although some investigations denoted contradictory results ^[29]. In addition to the already mentioned processes, several studies have determined effects of quercetin in different specific mechanisms ^{[26][32][44][45][46]}. These include inhibition of chymotrypsin-like activity of the proteasome, involved in proteasomal regulation of cancer signaling pathways ^[26][32]; rise of intracellular labile zinc, which has second messenger molecule activities in tumor cells ^[46]; and modulation of microRNAs expression ^[45]. Curiously, another study published in 2018 employed quercetin to analyze adequacy of the cellular antioxidant (CAA) assay in HepG2 cells in order to determine the antioxidant activity of extracts from tree nuts ^[44]. Besides, beneficial effects of quercetin against HCC were evaluated focusing on the sensitization of chemoresistant liver cancer cells and its hepatoprotective effects ^{[25][31][47][48]}.

Altogether, the wide variety of antitumor effects of quercetin along with its demonstrated efficacy against HCC cells, set this flavonoid as a promising therapeutic agent in the treatment of HCC.

3. Encapsulation strategy for the improvement of quercetin effects in HCC

Drug delivery systems have emerged as a novel mechanism of targeting cancer progression, enhancing drug efficacy through its encapsulation ^[49]. In this line, nanomedicine has developed numerous nanoparticles employing either organic- or inorganic-based nanocarriers ^[50]. Mesoporous silica nanoparticles (MSNs) conjugated with folic acid (FA) were designed to improve antitumor activity of quercetin. As inorganic-based carriers, gold-nanoparticles are drug delivery systems commonly chosen in nanomedicine ^[50]. Different publications synthesized poly(DL-lactide-co-glycolide) (PLGA)-loaded gold-quercetin nanoparticles leading to higher antiproliferative actions of quercetin in different HCC cell lines and in a xenograft tumor mouse model ^[49][51]. Otherwise, changes in cell cycle modulation were also observed after quercetin-nanocapsules administration and apoptosis induction was incremented in both in vitro and in vivo experiments ^[49][51]. In addition to cell proliferation and apoptosis, PLGA nanoparticles carrying quercetin generated morphologic alterations and inhibited migration ability of HCC cells ^[49][51]. Likewise, another group of researchers demonstrated greater cell death stimulation by encapsulating quercetin into PLGA nano-prototypes decorated with chitosan (CS) and polyethylene glycol (PEG) in HepG2 cells ^[52].

Some studies have also evaluated lipid-based formulations as quercetin-encapsulation strategies ^[53][54]. Solid lipid nanoparticles (SLNs) containing three sterol variables (cholesterol, stigmastanol and stigmasterol) were designed and evaluated, rising quercetin-derived cell viability reduction ^[54]. Similarly, the employment of methoxy-poly(ethylene glycol)-b-oligo(e-caprolactone), mPEG750-b-OCL-Bz micelles to co-encapsulate quercetin and superparamagnetic iron oxide nanoparticles (SPIONs) denoted lower proliferative capacity, morphological changes and cell cycle arrest in G0/G1 phase in an HCC model ^[53].

Even though few studies analyzed quercetin nanoencapsulation as a drug delivery system in liver cancer cells, it has arisen as a novel therapeutic approach which could improve quercetin properties against HCC progression by specifically targeting tumor and increasing drug cellular uptake.

4. Synergistic effects through quercetin combination against HCC 

Although a great number of antitumor properties of quercetin have been established in HCC treatment mainly in cell line but also in animal models, some researchers have focused on searching for synergistic combinations with this flavonoid with the aim of improving its effectiveness against liver cancer ^{[33][34][35][40][36][37][38][39][55][56]}. Enhanced properties with the well-stablished first-line drug sorafenib was demonstrated in several HCC cell lines by reducing its half-maximal inhibitory concentration (IC50) ^[36] and improving its tumor suppression activity ^[33]. Similarly, quercetin was able to raise antiproliferative action of several molecules, such as interferon-a (IFN-a) ^[34]; an oncolytic adenovirus expressing tumor necrosis factor-related apoptosis inducing ligand (ZD55-TRAIL) ^[56]; two derivatives of the organic compound maleic anhydride (3’5’-dimaleamylbenzoic acid and 3’5’-dimaleimylbenzoic acid) ^[35]; the chemotherapeutic drugs celecoxib ^[40], 5-fluorouracil (5-FU) ^[37] and cisplatin ^[38]; and the CDK inhibitor roscovitine ^[39]. Results reported quercetin-derived alterations in cell morphology when combined with roscovitine ^[39] as well as growth inhibition and cell cycle arrest after cisplatin and two maleic anhydride derivatives co-administration ^[35][38]. A higher quercetin-associated apoptosis was also observed when administered with the following compounds, ZD55-TRAIL ^[56], two maleic anhydride derivatives ^[35], celecoxib ^[40], cisplatin ^[38] and roscovitine ^[39].

Regulation of oxidative stress and ROS production by quercetin was also altered after addition of 3’5’-dimaleamylbenzoic acid and 3’5’-dimaleimylbenzoic acid, showing an increase in the reactive oxygen species (ROS) accumulation ^[35]. On the other hand, effects of quercetin and dasatinib combination in cell senescence were assessed in HCC cells, which showed inability to prevent doxorubicin-induced senescence ^[55].

It should be mentioned that two independent studies put together both quercetin combination and encapsulation strategies and evaluated its cytotoxic actions both in vitro and in vivo models ^[42][57]. Lactoferrin shell-oily core nanocapsules coupled with lactobionic acid (LA) or glycyrrhetinic acid (GA) were designed for targeted delivery of both quercetin and sorafenib, showing greater antitumor effects in HepG2 cell line and HCC-bearing mice ^[42]. Similar results were described with arginine-glycineaspartic acid (RGD)-modified lipid-coated nanoparticles loaded with quercetin and sorafenib using HepG2 cells and a mouse model of HCC ^[57].

Globally, co-treatment strategy of quercetin with different compounds may enhance its effectiveness by raising mainly its antiproliferative and proapoptotic effects and leading to improve quercetin single-therapy properties against HCC.

5. Effects of quercetin derivatives as treatment in HCC

Quercetin glycosides are one of the main quercetin naturally occurring forms, what makes them interesting compounds for cancer treatment ^[58]. This led several researchers to analyze effects of quercetin derivatives in different tumors, including HCC ^{[43][58][59][60]}. In this line, quercetin-3-O-glucoside (Q3G) and six long chain fatty acid esters of Q3G (stearic acid ester, oleic acid ester, linoleic acid ester, alpha-linoleic acid ester, eicosapentaenoic acid ester and docosahexanoic acid ester) displayed antiproliferative actions, cell cycle arrest and apoptosis induction in in vitro models of HCC ^[58][60]. On the other hand, 3,4-dihydroxyphenylacetic acid (DOPAC), a catabolite of some quercetin glycosides produced by colonic microflora, has been shown to modulate oxidative stress enzymes and to protect against acetaldehyde damage ^[59]. In the same way, both 3’-O-methyl quercetin (3’MQ) and quercetin-3-O-glucuronide (Q3GA) exhibited antioxidant properties by reverting ethanol-derived ROS accumulation ^[43].

Bioactive compounds derived from quercetin have been shown to abrogate cancer progression in liver cancer cells; nonetheless, a greater number of studies would be needed to search for more quercetin derivatives and study underlaying mechanisms of its antitumor action against HCC cells.

Main findings here described reporting quercetin effects in both in vitro and in vivo models are summarized in Table 1 and Table 2, respectively.

Table 1. Basic characteristics of in vitro studies using quercetin in single, encapsulated, combined or derived forms in HCC.

¹⁸F-FDG: fluorine-18 fluorodeoxy-glucose; 2’5’-OAS: 2’5’ oligoadenylate synthetase; 2-DG: 2-deoxy-D-glucose; 3’MQ: 3’-O-methyl quercetin; 4-HNE: 4-hydroxynonenal; 4EBP1: eukaryotic translation initiation factor 4E-binding protein 1; 5-FU: 5-fluorouracil; ABCB1: ATP-binding cassette subfamily B member 1; ABCC1: ATP-binding cassette subfamily C member 1; ABCC2: ATP-binding cassette subfamily C member 2; ABTS.+: radical cations of 2,2’-azino-bis(3-ethyl-benzothiazoline-6-sulphonic acid) diammonium salt; ADR: doxorubicin; AFB₁: aflatoxin B₁; AhR: aryl hydrocarbon receptor; Akt: protein kinase B; ALDH: aldehyde dehydrogenase; ALDH1A1: aldehyde dehydrogenase 1 member A1; ALDH2: aldehyde dehydrogenase 2; ALDH3A1: aldehyde dehydrogenase 3 member A1; AP-1: transcription factor AP-1; Apaf1: apoptotic protease-activating factor 1; B(a)P: benzo[a]pyrene; Bad: Bcl-2-associated agonist of cell death; Bax: Bcl-2-associated X; Bcl-xL: Bcl-2-like protein 1; Bid: BH3-interacting domain death agonist; CAA: cellular antioxidant activity; CAT: catalase; CDK1: cyclin-dependent kinase 1; CHK1: checkpoint kinase 1; COX-2: cyclooxygenase-2; CS: chitosan; DOPAC: 3,4-dihydroxyphenylacetic acid; ERK1/2: extracellular signal-regulated kinase 1/2; FA: folic acid; FLIP: FLICE-like inhibitory protein; FZD7: Frizzled homolog protein 7; GA: glycyrrhetinic acid; Gadd45b: growth arrest and DNA damage-inducible protein GADD45 beta; GLUT-1: glucose transporter type 1; GSH: glutathione; GSSG: oxidized glutathione; HCC: hepatocarcinoma; HDAC: histone deacetylase; HK2: hexokinase-2; HO-1: heme oxygenase-1; hTERT: telomerase reverse transcriptase; IC50: half-maximal inhibitory concentration; IFN-a: interferon-a; IGF2BP1: insulin-like growth factor-2 binding protein 1; IGF2BP3: insulin-like growth factor-2 binding protein 3; IkBa: nuclear factor-kB inhibitor a; IKKa: inhibitor of nuclear factor-kB kinase subunit a; IL-6: interleukin 6; ISRE: interferon-sensitive response element; Jak1: Janus kinase 1; JNK: c-Jun N-terminal kinase; LA: lactobionic acid; LC3: microtubule-associated protein 1 light chain 3; LC3A: microtubule-associated protein 1A/1B light chain 3A; LC3B-II: microtubule-associated protein 1A/1B light chain 3B; LF: lactoferrin; MDA: malondialdehyde; Mcl-1: induced myeloid leukemia cell differentiation protein; MMC: mitomycin; MMP-7: matrix metalloproteinase-7; MMP-9: matrix metalloproteinase-9; MSNs: mesoporous silica nanoparticles; mTOR: mammalian target of rapamycin; NF-kB: nuclear factor-kB; Nrf2: nuclear factor erythroid 2-related factor 2; p38 MAPK: mitogen-activated protein kinase p38; p62: sequestosome-1; p70S6K: ribosomal protein S6 kinase beta-1; PARP: poly(ADP-ribose) polymerase; PCNA: proliferating cell nuclear antigen; PEG: polyethylene glycol; PIG3: p53-inducible gene 3; PKC: protein kinase C; PKR: RNA-activated protein kinase; PLGA: poly(DL-lactide-co-glycolide); Q3G: quercetin-3-O-glucoside; Q3GA: quercetin-3-O-glucuronide; RGD: arginine-glycineaspartic acid; Rh123: rhodamine 123; ROS: reactive oxygen species; SHP-1: Src homology domain 2 tyrosine phosphatase-1; SHP-2: Src homology domain 2 containing tyrosine phosphatase-2; SLNs: solid lipid nanoparticles; SOD: superoxide dismutase; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; Sp1: specificity protein 1; SPION: superparamagnetic iron oxide nanoparticles; STAT1: signal transducer and activator of transcription 1; STAT3: signal transducer and activator of transcription 3; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; Tyk2: non-receptor tyrosine-protein kinase 2; ZD55-TRAIL: oncolytic adenovirus expressing tumor necrosis factor-related apoptosis inducing ligand.

Table 2. Basic characteristics of in vivo studies using quercetin in single, encapsulated, combined or derived forms in HCC.

5-FU: 5-fluorouracil; Akt: protein kinase B; ALT: alanine aminotransferase; AST: aspartate aminotransferase; Bax: Bcl-2-associated X; COX-2: cyclooxygenase-2; DEN: diethylnitrosamine; ERK1/2: extracellular signal-regulated kinase 1/2; GA: glycyrrhetinic acid; HCC: hepatocarcinoma; HK2: hexokinase-2; hTERT: telomerase reverse transcriptase; IkBa: nuclear factor-kB inhibitor a; IKKa: inhibitor of nuclear factor-kB kinase subunit a; LA: lactobionic acid; LC3A: microtubule-associated protein 1A/1B light chain 3A; LC3B: microtubule-associated protein 1A/1B light chain 3B; LF: lactoferrin; mTOR: mammalian target of rapamycin; NF-kB: nuclear factor-kB; p62: sequestosome-1; PCNA: proliferating cell nuclear antigen; PLGA: poly(DL-lactide-co-glycolide); RGD: arginine-glycineaspartic acid; RLW: relative liver weight; TNF-a: tumor necrosis factor-a ; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VEGF: vascular endothelial growth factor; ZD55-TRAIL: oncolytic adenovirus expressing tumor necrosis factor-related apoptosis inducing ligand.

6. Conclusion

In conclusion, quercetin seems to have a clear antiproliferative and proapoptotic effect against HCC, and likely a modulating role on tumor cell cycle progression which needs to be deeper investigated. This flavonoid seems to have antitumoral efficacy through the alteration of a great variety of cellular processes and signaling pathways, though more studies are required to further elucidate its mechanisms of action against HCC. Arising strategies of combination and drug-delivery systems may improve such cancer inhibition properties and, along with emergent use of quercetin derivatives with anticancer efficacy, let enlarging therapeutic options for HCC patients.

The entry is from 10.3390/nu11122875