Therapeutic effects of quercetin in oral cancer therapy: a systematic review of preclinical evidence focused on oxidative damage, apoptosis and anti-metastasis

Abstract

Objective

Oral malignancies are among the common head and neck cancers. Various therapeutic modalities are used for targeting oral cancers. It was shown that quercetin (a flavonoid) has an anti-cancer effect on different cancers. In the current study, the anti-cancer potentials of quercetin against oral cancer cells were summarized.

Methods

The current systematic review was conducted in accordance with the PRISMA guideline for the identification of relevant studies in various electronic databases up to April 2023. After reviewing and screening 193 articles, 18 were chosen for this study based on our inclusion and exclusion criteria.

Results

It was shown that quercetin significantly reduced cancer cell proliferation, cell viability, tumor volume, invasion, metastasis and migration. This anti-cancer agent induced oxidative stress and apoptosis in the cancer cells. Quercetin treatment could also induce some biochemical alterations in the cancer cells.

Conclusion

According to the results, it can be mentioned that quercetin administration has an anti-cancer effect against oral cancer cells. This agent exerts its anticancer effects via reduced cell viability and different mechanisms, including induce oxidative damage, apoptosis, and reduced invasion and metastasis. However, suggesting the use of quercetin as a therapeutic agent of oral cancer patients requires further clinical studies due to its poor absorption rates, and the exact molecular mechanisms are still not well understood.

Introduction

Cancer is the leading cause of morbidity and death worldwide and is regarded as one of the most significant health issues that imposes high expenses on society. Oral cancer is the sixth most prevalent cancer in the globe, which develops in the lip, oral cavity, and oropharynx [1–4]. The main predisposing risk factors that contribute to the etiology of oral cancer include heavy alcohol consumption, tobacco, pre-malignant conditions, poor oral hygiene, increased age, and viral infections such as human papillomavirus and Epstein-Barr virus [5, 6]. Even though the relationship between the abovementioned risk factors and oral cancer has been established, further research is necessary [7]. However, treatment strategies for this disease and these symptoms continue to be inadequate, which can be attributed to the phenotypic changes accompanying aging [7]. There are multiple treatments available for oral cancer, including biological therapies, surgery, hormone therapy, radiation therapy, chemotherapy, cryotherapy, molecularly targeted medications, and immune checkpoint inhibitors. In addition to these therapeutic modalities, advanced oral cancer can be treated with adjuvant therapy [8, 9].

During the past years, the tendency to use the natural compounds derived from plants for treating various cancers has attracted much attention [10–15]. In recent years, there has been increasing interest in potential effects of quercetin in cancer prevention and treatment [16, 17]. Quercetin is a flavonoid found in many vegetables, grains, seeds, leaves, and fruits [18, 19]. This herbal agent has been shown to exert various anti-cancer properties. It has been reported, for instance, that quercetin inhibits the proliferation of cancerous cells, induces apoptosis in cancerous cells, and prevents the spread of cancerous cells to other parts of the body [20]. Quercetin, through its antioxidant properties, may also play a role in preventing and treating cancers, as it can neutralize free radicals in the body, thereby preventing oxidative damage to normal cells and cancer development [21, 22]. Besides its anti-cancer role, quercetin has been observed to exhibit neuroprotective effects in Alzheimer's disease through its anti-oxidants, and anti-inflammatory properties and inhibition of amyloid-β (Aβ) fibril formation [23, 24]. Additionally, quercetin protects the heart by stopping oxidative stress, inflammation, apoptosis, and protein kinases [25].

To the best of our knowledge, this study represents the initial comprehensive evaluation of the anti-cancer properties of quercetin on oral cancer cells through a systematic review. The present discourse aims to address the following subjects: Firstly, what is the mechanism behind the anti-cancer effects of quercetin? Secondly, what are the mechanisms through which quercetin exerts anti-cancer effects on oral cancer cells?

Methodology

We conducted a comprehensive and systematic search based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. For structuring the review process, we also designed a PICO framework [26] as follows: Participants (P): oral cancer cells (in-vitro) /oral cancer animals (in-vivo), Intervention (I): in-vivo and in-vitro studies of oral cancer treated with quercetin, Comparison (C): in-vivo and in-vitro studies of oral cancer without any treatment, and Outcomes (O): findings obtained from quercetin treatment of in-vivo and in-vitro studies of oral cancer compared to the untreated group.

Search strategy

A comprehensive and systematic search was done for obtaining all relevant scientific papers on “anti-cancer effects of quercetin in oral cancer therapy” in various online databases of PubMed/Medline, Scopus, and Web of Sciences. The search terms were selected based on the objectives of the present review: (Quercetin OR Dikvertin) AND (“Squamous Cell Carcinoma of Head and Neck” OR “oral Squamous Cell Carcinoma” OR “Head And Neck Squamous Cell Carcinomas” OR “Squamous Cell Carcinoma of the Head and Neck” OR “Head and Neck Squamous Cell Carcinoma” OR “Squamous Cell Carcinoma of the Larynx” OR “Laryngeal Squamous Cell Carcinoma” OR “Squamous Cell Carcinoma of Larynx” OR “Squamous Cell Carcinoma of the Nasal Cavity” OR “Oral Tongue Squamous Cell Carcinoma” OR “Hypopharyngeal Squamous Cell Carcinoma” OR “Oral Squamous Cell Carcinoma” OR “Oral Cavity Squamous Cell Carcinoma” OR “Oral Squamous Cell Carcinomas” OR “Squamous Cell Carcinoma of the Mouth” OR “Oropharyngeal Squamous Cell Carcinoma” OR “Mouth Neoplasms” OR “Mouth Neoplasm” OR “Oral Neoplasm” OR “Oral Neoplasms” OR “Cancer of Mouth” OR “Mouth Cancers” OR “Oral Cancer” OR “Oral Cancers” OR “Cancer of the Mouth” OR “Mouth Cancer” OR “oral cavity cancer” OR “oral carcinogenesis”) until April 2023.

Study selection

Initially, all publications were chosen according to the objective of the current study (i.e., the anti-cancer effects of quercetin in oral cancer therapy) as indicated in the title and abstract. Then, we included the publications with adequate results, English language, and no limitation on publication year in this systematic review. We also excluded case reports, book chapters, review articles, oral communications, posters, letters to the editors from the present systematic review.

Data collection

Two authors (MJS and HHA) independently extracted the following data from the eligible studies: author name, publication year, models (in-vivo or/and in-vitro studies), protocol, dosage and administration route of quercetin, and outcomes of quercetin co-administration.

Quality assessment

The quality assessment method used in this review was a modified version of Risk of Bias (RoB) 2, which was taken from a previous study that looked at in-vitro research [27]. Moreover, the SYRCLE risk of bias tool for animal studies was employed to assess the quality of in-vivo studies. SYRCLE's Risk of Bias tool consists of 10 questions [28]. Two authors (FA and BF) individually examined all papers for each quality assessment criterion. Conflicts between these both authors were settled through dialogue.

Results

Search results

The preliminary search of the specified electronic databases produced 193 articles, of which 87 were duplicates and subsequently removed. Out of the remaining 106 publications, 57 were excluded after evaluating their titles and abstracts. Forty-nine papers were deemed suitable for full-text evaluation, whereas thirty-one articles were excluded. Ultimately, 18 articles were deemed eligible and incorporated into the current study. Figure 1 depicts the search methodology flowchart.

Fig. 1.

Flowchart illustrating the selection procedure employed in the current investigation

Quality assessment and risk of bias

All 17 in-vitro studies earned a “low risk” score when evaluated according to the previously established quality evaluation criteria (Table 1). In two studies [40, 41], there was no information on duplicates or repeats, so it was thought that this was enough to give the studies a low overall bias score. The quality assessment utilizing the SYRCLE risk of bias tool for three in-vivo studies is presented in Table 2. For questions 1, 3, 4, and 6, the response was “yes”; for question 2, the response was “no”; for questions 5, 7, 9, and 10, the responses were “unclear”; and for question 8, two responses were “yes”.

Table 1.

Summary of quality assessment of included in-vitro studies

Table 2.

Quality assessment according to the SYRCLE Risk of Bias tool

Data extraction

Table 3 enumerates the data obtained from each qualifying article by MJS and HHA. Disagreements between these two authors were settled through dialogue.

Table 3.

The characteristics of included studies

Author and year	Models (in-vitro or in-vivo)	Quercetin agents dosage/Route of administration/Treatment time	Outcomes of quercetin administration
Son et al. (2023) [29]	in-vitro (YD10B cells)	25μM & 50 μM / 48h	Cell viability: ↓Cell viability Cell cycle distribution: ↑SubG0/G1, ↑G1, ↑S, ↓G2/M Molecular markers: ↑Apoptosis, ↓Expression of Bcl-2, ↑ Cleavage of PARP, ↑Phosphorylated p38, ↑Activation of MAPK
	in-vitro (YD38 cells)	50 μM & 100 μM / 48h	Cell viability: ↓Cell viability Molecular markers: ↑SubG0/G1, ↑G1, ↓S, ↑G2/M ↑Apoptosis, ↓Expression of Bcl-2 (anti-apoptotic), ↑ Cleavage of PARP, ↑Phosphorylated p38
Chen et al. (2021) [30]	in-vitro (CAL-27 cells)	80 μM/24h	Cell viability: ↓Cell counting, Molecular markers: ↓CD36 protein, ↑ Levels of miR-1254
Chun-Fahuang et al. (2022) [31]	in-vitro (SAS cells)	50 µM/6 or 24 h	Cell viability: ↓Cell viability Molecular markers: ↑Apoptosis, ↑Caspase‑3 activity, ↓MMP, ↑Cytosolic Cyt. C, ↑ Bax & Bak protein expression, ↓Bcl‑2 protein expression, ↑Cleaved caspase‑3, caspase‑7, PARP, ↑ p‑ERK1/2, p‑JNK1/2, & p‑GSK3‑α/β
Kim et al. (2020) [32]	in-vitro (OSC20 cells)	40 µM/24h	Cell viability: ↓Cell Viability Cell cycle distribution: ↓Arrested G2 Phase Cell Cycle, Invasion: ↓Migration, ↑Expression of epithelial markers (E-cadherin & claudin-1), ↑Expression of mesenchymal markers (fibronectin, vimentin, & α-SMA), ↓Activation MMP-2 & MMP-9, ↓Expression levels of EMT inducers & MMPs, Downregulated Twist & Slug
	in-vitro (SAS cells)	40 µM/24h	Cell viability: ↓Cell Viability Cell cycle distribution: Arrested G2 Phase Cell Cycle Invasion: ↓Migration, ↑Expression of epithelial markers (E-cadherin & claudin-1), Expression of mesenchymal markers, (fibronectin, vimentin, & α-SMA), ↓Activation MMP-2 & MMP-9, ↓Expression levels of EMT inducers & MMPs, Downregulation of Twist & Slug, Induced translocation of Twist & Slug
	in-vitro (HN22 cells)	40 µM/24h	Cell viability: ↓Cell Viability Cell cycle distribution: Arrested G2 Phase Cell Cycle Invasion: ↓Migration, ↑Expression of epithelial markers (E-cadherin & claudin-1), Expression of mesenchymal markers, (fibronectin, vimentin, & α-SMA), ↓Activation MMP-2 & MMP-9, ↓Expression levels of EMT inducers & MMPs, Downregulation & inhibition of Slug activation
Zhao et al. (2019) [33]	in-vitro (HSC-6 cells)	50μM/48h	Cell viability: ↓Cell viability, Invasion: ↓Migration & Invasion, ↓MMP-9 & MMP-2 protein, ↑miR-16
	in-vitro (SCC-9 cells)	50μM/48h	Cell viability: ↓Cell viability Invasion: ↓Migration & Invasion, ↓MMP-9 & MMP-2 protein, ↑miR-16
Zhang et al. (2019) [34]	in-vitro (Tca8113 cells)	0μM, 25μM, 50μM, 100μM/24h	Cell viability: ↓Cell viability Molecular markers: ↑Apoptosis, ↓CC50, ↑Expression of miR-22, ↓ Activation of WNT1 & β -catenin, ↓Xenograft tumor growth
	in-vitro (SAS cells)	0μM, 25μM, 50μM, 100μM/24h	Cell viability: ↓Cell viability Molecular markers: ↑Apoptosis, ↓CC50, ↑Expression of miR-22, Activation of WNT1 & β -catenin, ↓Xenograft tumor growth
Li et al. (2019) [44]	in-vitro (Tca-8113 cells)	100μM/2h	Molecular markers: ↑Cytosolic IκBa, ↓Nuclear P65, ↓Activation of IKKβ & Akt
	in-vitro (SCC-15 cells)	100μM/2h	Molecular markers: ↑Cytosolic IκBa, ↓Nuclear P65, ↓Activation of IKKβ & Akt, ↓Cytosolic & Nuclear xIAP protein level
	in-vivo (nude mice)	50 mg/kg	Tumor weight: ↓Tumor weight Molecular markers: ↓ Activation of caspase-3, ↓ xIAP protein level, ↑PARP protein level
Ma et al. (2018) [35]	in-vitro (SAS cells)	40 µM /48 h	Cell viability: ↓Cell viability Molecular markers: ↑Apoptosis, ↑ROS, ↑Ca2+ production, ↑Activities of caspase‑3, caspase‑8 & caspase‑9, ↑Expression of caspase-2, Bak, Bid, Bad, Cyt. C, Apaf-1, Endo G, AIF, & PARP, ↑Activation of caspase-3, caspase-4, caspase-6, caspase-7, caspase-8, caspase-9, TRAIL, Fas-L, Fas, FADD, ATF-6α, ATF-6β, XBP-1, IRE-1α, & GRP-78, ↓Expression of Bcl-2, Bcl-x, pro-caspase-3, ↑Mitochondrial release of Cyt. C, AIF, & Endo G
Yuan et al. (2015) [36]	in-vitro (KB/VCR cells)	100µM 24h	Cell cycle distribution: ↑G1 phase, ↓S phase Molecular markers: ↑Apoptosis, ↓Bcl-2 levels, ↑Bax levels, ↓Pro-caspase-3, ↑Cleavage caspase-3, ↓ Protein levels of P-gp, & P-gp Expression Invasion: ↓Growth inhibition rate, Inhibited Migration & Invasion
Droguett et al. (2015) [46]	in-vivo (mice)		Molecular markers: ↓PCNA immunoreactivity
Lai et al. (2013) [37]	in-vitro (SAS cells)	50μM/24h	Cell viability: ↓viable cells, Molecular markers: ↓Activation of MMP-9 & MMP-2, ↓Protein levels of MMP-2, -7, -9, -10, VEGF, iNOS, COX-2, uPA, PI3K, IKB-α, IKB-α/β, p-IKKα/β, FAK, SOS1, GRB2, MEKK3, MEKK7, ERK1/2, p-ERK1/2, JNK1/2, p38, p-p38, c-JUN, & p–c-JUN Invasion: ↓Migration & invasion
Huang et al. (2013) [38]	in-vitro (HSC-3 cells)	20μM/24h	Cell viability: ↓Cell growth, ↓Colony formation Cell cycle distribution: ↑Cell numbers in the G2/M phase Molecular markers: ↑Cleaved caspase 3, Activation of executioner caspase 3, ↑Apoptosis, ↓protein levels of p-Y1086-EGFR, p-Akt, ↑FOXO1 protein levels, ↓phosphorylation of cytoplasmic FOXO1, ↑Translocation of FOXO1 from cytoplasm to nucleus, ↑Nuclear translocation & transactivates of FOXO1, ↑Fas-L expression
	in-vitro (TW206 cells)	20μM/24h	Cell viability: ↓Cell growth Cell cycle distribution: ↑Cell numbers in the G2/M phase Molecular markers: ↑Fas-L expression & cleaved caspase 3, ↑Activation of caspase 3, ↑Apoptosis
	in-vivo (Mice xenograft (HSC-3))		Cell viability: ↓Tumor size, weight Molecular markers: ↑FOXO1 expressions
Chen et al. (2013) [43]	in-vitro (SCC-25 cells)	75µM/24h	Cell viability: ↓Cell viability, ↓Colony growth assays Cell cycle distribution: ↑G1 phase cells, ↓S phase cells Molecular markers: ↑Apoptosis, ↓Bcl-2, ↑Bax level, ↑Expressions & active form of caspase 3, ↑Cleaved PARP Invasion: ↓Migration & invasive
Kang et al. (2010) [39]	in-vitro (SCC-1483 cells)	80µM/24h	Cell viability: ↓Living cells Molecular markers: ↑Apoptosis Invasion: ↓Cell proliferation
Haghiac et al. (2009) [40]	in-vitro (SCC-9 cells)	50µM/48h	Cell viability: ↓Cell Growth Molecular markers: ↓DNA synthesis, ↑Cell damage, ↑LDH release, ↑Necrotic & apoptotic Cell cycle distribution: ↑Population in the S phase, ↓Cells in the G1 phase, ↑Population in the G2-M phase Invasion: ↓Proliferation
Browning et al. (2005) [42]	in-vitro (SCC-9 cells)	10µM/24h	Invasion: ↓Proliferation
ElAttar et al. (1999) [41]	in-vitro (SCC-25 cells)	100µM/24h	Cell viability: ↓Cell growth
Hu et al. (2023) (45)	in-vitro (CAL27 cells)	80µM/24h	Molecular markers: ↓Activities of HK, PK, & LDH, ↓Glycolysis, ↓Glucose uptake, ↓Lactate production, ↓Viability, ↓G3BP1, & YWHA2 protein levels

↑, Increase; ↓, Decease; &, and; ip, Intraperitoneal; po, Per os; PARP, Poly (ADP-ribose) polymerase; MAPK, mitogen activated protein kinase; MMP, mitochondrial membrane potential; Bcl-2, B cell lymphoma 2; Bax, BCL2-associated X protein; Bak, Bcl-2 homologs antagonist/killer; ERK1/2, Extracellular signal-regulated kinase 1 and 2; JNK1/2, c-Jun N-terminal protein kinase (JNK); GSK3‑α/β, Glycogen synthase kinases 3; α-SMA, alpha-smooth muscle actin; EMT, epithelial-mesenchymal transition; IκBa, inhibitor of nuclear factor kappa B; IKK-β, inhibitor of nuclear factor kappa-B; xIAP, X-linked inhibitor of apoptosis protein; PARP, poly ADP-ribose polymerase; ROS, reactive oxygen species; Ca2 + , Calcium; Bid, BH3 interacting-domain death; Bad, Bcl-2 associated agonist of cell death; Apaf-1, Apoptotic protease activating factor-1; Endo G, Endonuclease G; AIF, apoptosis-inducing factor; TRAIL, TNF-related apoptosis-inducing ligand; FADD, FAS-associated death domain; ATF-6α, Activating Transcription Factor 6 Alpha; ATF-6β, Activating Transcription Factor 6 Beta; XBP-1, X-Box Binding Protein 1; IRE-1α, Inositol-requiring enzyme 1 alpha; GRP-78, Glucose-Regulated Protein 78; Bcl-x, B-cell lymphoma-extra; Endo G, Endonuclease G; P-gp, P-glycoprotein; PCNA, Proliferating cell nuclear antigen; VEGF, Vascular endothelial growth factor; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; uPA, urokinase-type plasminogen activator; PI3K, phosphoinositide 3-kinase; IKB-α, NF-kappa-B inhibitor alpha; IKKα/β, IκB kinase α/β; FAK, focal adhesion kinase; SOS1, salt overly sensitive 1; GRB2, Growth factor receptor-bound protein 2; MEKK3, Mitogen-activated protein kinase kinase kinase 3; MEKK7, Mitogen-activated protein kinase kinase kinase 7; Cyt. C, cytochrome c; FOXO1, Forkhead box protein O1; LDH, lactate dehydrogenase; HK, hexokinase; PK, pyruvate kinase; WNT1, Wnt family member 1

The anti-cancer potentials of quercetin against oral cancer cells

Cell viability

The data from the in-vitro studies revealed that the cell viability was meaningfully reduced in a dose-dependent and time-dependent manner by quercetin treatment compared to the non-treated cells [29–41]. In a study of Zhang et al., Tca8113 and SAS cells were exposed to 0, 25, 50, and 100 µM of quercetin, and doses of 50 and 100 µM significantly reduced cell viability after 24 h of treatment [34]. Furthermore, in the study Zhao et al., the HSC-6 and SSC-9 cells treated with doses of 0, 25, 50, and 100 µM of quercetin, the results demonstrated quercetin significantly reduced cell viability in doses of 50 and 100 µM [33]. Also in another study, the OSC20, SAS, and HN22 cells were exposed to doses of 0, 10, 20, 40, 80, and 160 µM of quercetin. The results demonstrate that the doses of 40, 80, and 160 µM in OSC20, 80 and 160 µM in SAS, and 160 µM in HN22 significantly reduced cell viability [32].

Furthermore, it was revealed that quercetin administration significantly reduced the cell proliferation of oral cancer cells (SCC-1483 and SCC-9) in comparison with the untreated cancer cells [39, 42]. Moreover, it was demonstrated that quercetin significantly reduced SCC-25 viability and colony formation doses decently [43].

Tumor weight

The results of an in-vivo investigation showed that the administration of quercetin led to a reduction in the size, weight, and growth of the tumors that were xenografted onto nude mice as compared to the group that was not given any treatment [38, 44].

Cell cycle distribution

According to the findings of the cell cycle research performed on oral cancer cells, quercetin treatment was able to halt the progression of the cell cycle in some phases, which resulted in the inhibition of cell division and a reduction in cell viability [29, 32, 36, 38]. The administration of quercetin has been shown in a few trials to be capable of causing a cell cycle arrest in oral cancer patients who are in the G1 phase [29, 36, 40, 43]. An additional discovery demonstrated that the application of quercetin resulted in the halting of the cell cycle of oral cancer cells in the G2 phase [29, 32, 38, 40].

Molecular markers

According to the findings, it was found that quercetin treatment causes molecular changes in oral cancer cells (in-vitro and in-vivo). In this regard, it was shown that the level of reactive oxygen species (ROS), calcium (Ca²⁺), lactate dehydrogenase (LDH) release, Fas, Fas ligand (Fas-L), Fas-associated death domain (FADD), caspase-2, caspase-3, caspase-4, caspase-6, caspase-7, caspase-8, and caspase-9, and release of endonuclease G (Endo G), cytochrome c (Cyt. C)and apoptosis-inducing factor (AIF) from mitochondria to the cytoplasm significantly increased in the groups treated with quercetin than the control/untreated groups. Moreover, it is shown the activity of p38, c-Jun N-terminal kinase (JNK) 1/2, glycogen synthase kinase 3 (GSK3) α/β, extracellular signal-regulated kinase (ERK) 1/2, poly ADP-ribose polymerase (PARP), X-box binding protein 1 (XBP-1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), activating transcription factor (ATF)-6α, ATF-6β, mitogen-activated protein kinase (MAPK), inositol-requiring enzyme 1 (IRE-1α), glucose-regulated protein-78 (GRP-78) meaningfully increase in compression of untreated group [29, 31, 35, 37, 38, 44, 45].

Moreover, quercetin significantly up-regulated Bcl-2-associated X protein (Bax), BH3 interacting-domain death agonist (Bid), Bcl-2-associated agonist of cell death (Bad), and Bcl-2 homolog antagonist/killer (Bak), apoptotic protease activating factor-1 (Apaf-1), epithelial markers (such as claudin-1 and E-cadherin), mesenchymal markers (such as vimentin, fibronectin, and alpha-smooth muscle actin (α-SMA)), nuclear translocation and transactivates of forkhead box protein O1 (FOXO1), and expression of miR-1254, miR-16, and miR-22 compared to control/untreated groups [30–38, 43].

In contrast, mitochondrial membrane potential (MMP), matrix metalloproteinase-2 (MMP-2), MMP-7, MMP-9 and MMP-10, B-cell lymphoma 2 (Bcl-2), LDH, hexokinase, pyruvate kinase, Vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), Wnt family member 1 (WNT1), β-catenin, phosphoinositide 3-kinase (PI3K), urokinase-type plasminogen activator (uPA), nuclear factor-kappa B inhibitor alpha (IκB-α), phosphor-IκB kinase (IKK) α/β, focal adhesion kinase (FAK), salt overly sensitive (SOS1), JNK1/2, mitogen-activated protein kinase kinase kinase 3 (MEKK3), growth factor receptor-bound protein 2 (GRB2), MEKK7, inhibitor of nuclear factor kappa-B (IKKβ), ERK1/2, p38, c-JUN, Akt, Y1086-EGFR, CD36, Bcl-extra (Bcl-x), caspase-3, epithelial-mesenchymal transition (EMT), P-glycoprotein (P-gp) levels significantly decreased in the groups treated with quercetin than the control/untreated groups. Moreover, quercetin meaningfully inhibited proliferating cell nuclear antigen (PCNA) immunoreactivity, reduced free thymidylate synthase (TS) protein, nuclear P65, cytosolic and nuclear X-linked inhibitor of apoptosis protein (xIAP), and glucose uptake, lactate production, as well as down-regulated protein levels of G3BP1, YWHA2, Twist, and Slug compared to the control and untreated groups. Quercetin could also induce the translocation of Twist protein from the cytosol to the nucleus and Slug from the nucleus to the cytosol [29–38, 40, 43–46].

Invasion

According to the results of four studies, it was found that quercetin treatment significantly reduced oral cancer cell migration and invasion. In the Yuan et al., study, it was demonstrated that quercetin at doses of 25, 50, and 100 μM significantly reduced migration and invasion in KH/VCR cells [36]. Moreover, in the Zhang et al., study, quercetin at a dose of 50 μM remarkably reduced HSC-29 and SCC-9 migration and invasion [34]. In another study, quercetin at doses of 25 and 50 μM remarkably reduced migration and invasion in SAS cells [33]. Also, in another study quercetin minigfully in dose dependent reduced migration and invasion of SCC-25 cancer cells in doses of 25, 50, and 75 μM [43].

Discussion

This systematic study elucidates the anti-cancer efficacy of quercetin in the treatment of oral cancer. Quercetin exerts anti-cancer benefits through many mechanisms, including the enhancement of oxidative stress and apoptosis, as well as the inhibition of metastasis, and angiogenesis [47–49]. The action mechanisms of quercetin may be attributed to its influence on mitochondrial function, modification of tumorigenic gene expression, transcription factors, and related factors [50–52]. Figure 2 depicts the primary anti-cancer mechanisms of quercetin in the treatment of oral cancer. The subsequent discussion will address several of these mechanisms.

Fig. 2.

Depicts the essential pathways that underpin the anti-cancer properties of quercetin in relation to oral cancer. Quercetin works by increasing the levels of cytosolic Ca²⁺, decreasing the levels of anti-apoptotic proteins (Bcl-2 and Bcl-xL), and raising the levels of pro-apoptotic proteins (Bid, Bad, and Bak). This causes Cyt.C levels to rise in the cytosol and the apoptosome complex to form. This then activates caspase-3 and finally leads to apoptosis. ↑ increased by quercetin; ↓ decreased by quercetin; reactive oxygen species (ROS), apoptotic protease activating factor 1 (Apaf-1), cytochrome C (Cyt. c), nuclear factor kappa-β (NF-κB), inhibitor of nuclear factor kappa B (IKB), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), BH3 Interacting Domain Death (Bid), BCL2 associated agonist of cell death (Bad), BCL-2 antagonist/killer (Bak)

It has been demonstrated that quercetin, through several ways, could reduce cell viability, proliferation, and invasion of oral cancer cells. In this case, it is reported that miR-1254 controls the expression of CD36 (a glycosylated membrane protein), which is linked to the growth and development of cancer. One way quercetin might stop oral cancer cells from spreading is by decreasing the amount of CD36 and increasing the amount of miR-1254 [30]. Moreover, TGF-β1 induces EMT in diverse cancer cells, thereby promoting their motility and invasiveness [53]. Several transcription factors, including Twist, Snail, and Slug, are able to down-regulate E-cadherin (a tumor suppressor protein) [29, 54]. TGF-β1 can down-regulate Twist1 and Snail1, as well as reduce the expression of E-cadherin [54]. Furthermore, it has been shown that MMP-2 and MMP-9 are involved in invasion, cell migration, and the production of TGF-β1 (an immunomodulatory protein) and VEGF and angiopoietin-2, which are angiogenic factors [55, 56]. In a study conducted by Son et al., it was observed that quercetin exhibited the ability to decrease the activity of MMP-2 and MMP-9 enzymes. This reduction in enzyme activity resulted in a significant decrease in the migration and invasion of cells in oral cancers. Furthermore, quercetin was found to regulate the expression of genes associated with EMT, thereby influencing the cellular processes involved in cancer progression [29]. In a study conducted by Son et al., it was observed that quercetin exhibited the ability to decrease the activity of MMP-2 and MMP-9 enzymes. This reduction in enzyme activity resulted in a significant decrease in the migration and invasion of cells in oral cancers. Furthermore, quercetin was found to regulate the expression of genes associated with EMT, thereby influencing the cellular processes involved in cancer progression.

Vimentin is an intermediate filament that is utilized to distinguish mesenchymal cells from epithelial cells [57]. Vimentin is expressed at sites of cellular elongation and is linked to a phenotype associated with migration [58]. In other words, vimentin up-regulation is often used as a marker of EMT in cancer [59, 60]. In normal epithelial cells, the α-SMA protein promotes wound healing and embryogenesis [61]. Growth factors and specialized extracellular matrix (ECM) proteins control α-SMA expression. In addition, it is reported that α-SMA has a significant role in the development and progression of different solid tumors [62]. In oral cancer cells, quercetin could inhibit EMT via up-regulation of claudin-1 and E-cadherin and down-regulation of α-SMA, vimentin, fibronectin, and Slug [29]. It is shown that miR-22 significantly attenuates cell proliferation, migration, invasion, and EMT and also induces cell apoptosis in several cancers [34]. In this regard, miR-22 is able to impede the aforementioned effect by targeting snail and MAPK 1 [63, 64]. Moreover, it has been demonstrated that Wnt1 expression is associated with cancer proliferation [65]. Quercetin could inhibit cancer cell viability, proliferation, migration, and invasion through modulation of the miR-22/Wnt1/β-catenin pathway in oral cancers (both in-vivo and in-vitro experiments) [34].

The absence of equilibrium between the production of free radical agents and the body's anti-oxidant defenses is defined as oxidative stress [66, 67]. In a normal cell, oxygen radicals (O²⁻) are produced when electrons escape from the mitochondria's electron transport chain (ETC). The rapid coupling of the OH⁻ and O²⁻ radicals with the NO radical produces ONOO⁻ (a powerful reactive nitrogen species) [68]. In normal cells, quercetin could reduce ROS generation [69]. What is surprising is that it has been demonstrated that quercetin treatment elevates ROS generation in oral cancer cells [35]. It is established that oxidative stress is related to apoptosis, inflammation, aging, and other mechanisms [70].

The association between apoptosis and oxidative stress is intricate and complicated. ROS have the ability to stimulate caspases, which are a collection of enzymes that have a crucial function in the process of apoptosis [71, 72]. Furthermore, ROS has the ability to indirectly induce apoptosis by causing harm to cellular components and initiating stress signaling pathways. In addition, oxidative stress can result in the buildup of malfunctioning mitochondria, which can release Cyt. C, a protein that triggers apoptosis [73, 74].

The results shown in Table 1 show that quercetin treatment causes apoptosis in oral cancer cells through a mitochondrial-dependent pathway and increases the levels or activation of proteins such as TRAIL, FADD, Fas, Fas-L, and caspase-8 [35, 38]. Moreover, it is demonstrated that quercetin administration leads to the opening of the mitochondrial permeability transition pore (mPTP) through an increase in the activity of pro-apoptosis proteins (Bid, Bad, and Bak) and a reduction in the activity of anti-apoptotic proteins (Bcl-x and Bcl-2) [29, 31, 35–37]. Due to the opening of mPTP, Cyt. C releases into the cytosol. After that, Cyt. C joins with caspase-9 and Apaf-1 to produce the apoptosome, thereby activating caspase-3. Caspase-3 causes cells to commit suicide by cleaving the pro-apoptotic protein PARP and activating the deoxyribonuclease DNase [75]. Quercetin, through modulating the activity of the aforementioned proteins, can induce apoptotic death in oral cancer cells [29, 31, 35, 37, 44]. In more detail, quercetin induced an increase in pro-apoptotic proteins and a decrease in anti-apoptotic proteins. Furthermore, the mitochondrial release (induced by quercetin) increased amounts of Endo G, AIF, and Cyt. C into the cytoplasm of oral cancer cells. After that, there was more formation of the caspase-9/Apaf-1 apoptosome, which caused caspase-3 to become active and eventually led to apoptosis [31, 35, 36].

Endoplasmic reticulum (ER) stress releases cytosolic Ca²⁺ and activates the unfolded protein response (UPR) pathway [76, 77]. Inositol-requiring protein-1 (IRE-1), protein kinase RNA (PKR)-like ER kinase (PERK), and ATF6 are the main sensor proteins of UPR (as ER-resident integral membrane proteins) [78, 79]. Glucose-regulated protein (GRP)78/BiP is the main ER that is able to control the activation of the ER-transmembrane signaling molecules [80]. UPR pathways restore ER function if ER stress is too high; of note, the UPR mechanisms induce apoptosis through the intrinsic endoplasmic reticulum pathway [81]. Calpain-1 activates caspase-12 to induce apoptosis when it increases intracellular Ca²⁺ in ER stress [82]. Quercetin controls the activation of intracellular Ca²⁺ and calpain-1, which then activates GRP78, caspase-12, and C/EBP homologous protein (CHOP) in oral cancer cells [35, 83]. Moreover, the NF-κB pathway induces up-regulation of some genes, which inhibits apoptosis. It was shown quercetin treatment causes the elevation of ROS and activation of IκB, resulting in the activation and nucleus translocation of NF-κB [35, 44]. The activated NF-κB binds to certain gene promoters in the nucleus and eventually causes the expression of genes that cause cell death, such as Bcl-xL and A1/Bfl-1 [84, 85].

Furthermore, quercetin induces apoptosis through activation of p38, JNK, ERK, and NF-κB [29, 31]. The findings of the current systematic review also showed that quercetin-mediated apoptosis might occur as a result of elevated MAPK activation by PARP cleavage and p38 activation [29, 31, 35, 37, 44]. In addition, it has been hypothesized that JNK activation-mediated ERK/GSK3‑α/β signaling has a crucial role in the down-stream regulation of mitochondria-dependent apoptosis following quercetin treatment of oral cancer cells.

It is also shown that miR-16 directly regulates FEAT and cancer pathogenesis [86, 87]. miR-16 is highly expressed in normal tissues and is often deleted and upregulated in many types of cancer tissues [86]. Although miR-16 has many other targets, it has been found that it can lead to increased apoptosis in cancer cells through the downregulation of FEAT [86]. Quercetin was found to raise miR-16, which stops tumor growth and helps cells die. It could also cause apoptosis and stop oral cancer cells from spreading [33, 88].

The efficacy of quercetin (as lipophilic) is much impacted by its poor absorption rates, which define its bioavailability. The research on quercetin's bioavailability in animal models shows it may be as low as 10% [89]. In humans, variables like dietary composition (especially the presence of lipids that improve absorption) further complicate evaluations of bioavailability [90]. The recommended dosages for quercetin vary from 500 mg to 1 g per day [91]. The common reported of mild adverse effects of quercetin consumption in high dose included headaches, gastrointestinal issues such as gastro-esophageal reflux disease (GERD), stomach upset, and respiratory symptoms like breathlessness and chest congestion [91–93]. The lack of standardized dosing protocols hinders clinical application, as optimal dosages for specific conditions remain undetermined [94]. Also, even though preclinical studies have shown promise, the fact that disease models and treatment plans vary shows that the exact molecular mechanisms are still not well understood. This means that more research is needed to fully understand their effects.

Quercetin could be helpful in clinical settings for treating cancer. Ferry et al. found that quercetin was able to lower a patient's cisplatin dose from 295 units/ml to 55 units/ml. This patient had ovarian cancer and was resistant to cisplatin. They gave this patient two courses of 420 mg/m² of quercetin [95]. In addition, Kooshyar et al. did a controlled double-blind clinical trial study and found that quercetin could stop oral mucositis caused by chemotherapy. They also suggested that more studies be done with larger groups of people [96].

Conclusion

This systematic study revealed that quercetin therapy for oral cancer cells significantly reduced the viability, proliferation, growth, invasion, metastasis, and motility of the cancer cells. There are several ways that quercetin fights cancer. These mainly include the induction of oxidative stress, apoptosis, mitochondrial malfunction, and reduced invasion and metastasis. However, given that the results of the present study are derived from in-vitro and in-vivo experiments, the application of quercetin in oral cancer patients necessitates further clinical investigations, as the outcomes from experimental studies may not align with clinical results.

Author contributions

M.J.S., H.H.A., M.Ch., and A.F.A. gave the idea and drafted the manuscript. J.A.H., A.M., W.M.T., M.A., M.J.J., A.M.A.A., and R.A-S. performed the literature search and drafted figures. F.A. and B.F. edited the manuscript and supervised the whole study. All authors read and approved the manuscript.

Funding

None.

Availability of data and materials

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.