Cigarette smoke contains a large number of carcinogenic compounds, including nitrosamines (nitrosonornicotine, NNN; and 4-methylnitrosamino-1-3-pyridyl-1-butanone, NNK), which are implicated in increasing the risk of NSCLC [4]. Nicotine is not regarded as a carcinogenic compound in tobacco and cigarette smoke, but it has the potential to facilitate tumorigenesis [5,6]. Nicotine and NNK increase the NSCLC risk by upregulating a network of signaling pathways facilitating proliferation, metastasis, and angiogenesis, and inhibiting apoptosis through activation of the nicotinic acetylcholine receptor (α7nAChRs) and beta-adrenergic receptor (β-AdrR) [6,7,8]. Quercetin (Q) and its glycoside isoquercitrin (IQ) and rutin (RU) are natural flavonols found mostly in fruits, and are considered as positive allosteric modulators (PAMs)/selective agonists of human α7nAChR, which makes them potential therapeutic agents in nicotine-induced NSCLC [16,17]. 

2. Rutin

RU (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside), also known as Q-3-O-rutinoside, is a flavonol compound found in various fruits and vegetables. It is less bioavailable and soluble than Q in humans [31]. RU is also absorbed more slowly than Q in the small intestine of rats [32].

RU could be useful in the treatment of NSCLC mediated by α7nAChR. RU and Q act on α7nAChR-dependent ion currents. Q induces the enhancement of ACh-induced inward peak currents (I_ACh) on cells expressing human α7nAChR and increases the extracellular Ca²⁺ level-mediated potentiation of I_Ach via interactions with Ca²⁺-binding sites for α7nAChR in Xenopus oocytes (a system for the expression of plasma membrane transport proteins) [33]. Q exhibits differential regulation of α7nAChR channel activity with respect to RU in such a way that Q increases I_ACh, while RU decreases I_ACh in Xenopus oocytes expressing human α7AChR, and such effects exist in a voltage-insensitive and non-competitive manner. Q-mediated I_ACh suppression can be improved when Q is co-administered with RU, suggesting that RU may have a significant role in the regulation of α7nAChR [34].

RU was found to inhibit β-amyloid (Aβ) peptide-induced neuronal cytotoxicity, nitric oxide (NO), and the production of reactive oxygen species (ROS) and proinflammatory cytokines by reducing interleukin (IL)-1β and tumor necrosis factor (TNFα) production in microglia [35]. Plasma levels of Aβ were found to be significantly increased in patients with multiple cancers, including LC [36].

An in vitro study has shown that α-L-rhamnosidase (α-R) cleaves terminal α-rhamnose in flavonoid rutinosides/glycosides (hesperidin, naringin, diosmin, and troxerutin), which increases the anti-proliferative and anti-oxidant activities of RU against various cancer cell lines, including NSCLC (H460) cells [37]. In contrast, β-glucosidase (β-G) has been shown to exert high potency against LC tumor growth. β-G converts rutin to rutinose disaccharide and Q through its ability to remove sugar moieties such as glucose linked to flavonoids in positions 3 and 7 of the C ring [38]. β-G has been described as the key glycoprotein-processing enzyme that inhibits the expression of p53 in NSCLC cells [39]. Knockout of β-G was found to inhibit cell migration/metastasis and induce apoptosis and/or autophagy in NSCLC cells through the suppression of RTK signaling pathways [40].

α-R may convert RU into IQ with high bioactivity and bioavailability by combining with β-G [41]. Thus, α-R coupled with β-G provides high bioactive effects, which inhibit the proliferation and induction of apoptosis in NSCLC cells. Recent molecular docking studies showed that the OH groups of RU (C7-OH) form stable H-bond interactions with several amino acid residues, including serine, aspartic acid, phenylalanine, glutamine, glutamic acid, and arginine [42]. Therefore, these OH groups may create interactions with amino acid residues in the binding pocket of α7nAChR, displaying complex modulation of the receptor.

A few studies investigating the therapeutic efficacy of RU in nicotine-induced NSCLC showed that the mechanisms underlying its effects have not been explored. Transformation of rutin to quercetin-3-β-D-glucoside (Q3G) using α-R and β-G from a crude enzyme extract of Aspergillus niger (A. niger) has been shown to inhibit NSCLC cell proliferation [43]. A study has demonstrated that RU-loaded liquid crystalline nanoparticles exert anti-proliferative/anti-migratory and apoptotic effects on NSCLC cells [44]. RU reduces superoxide anion/intracellular ROS production and suppresses the proliferation and migration/adhesion of NSCLC cells, although it showed cytotoxic effects at concentrations higher than 500 µM (IC₅₀ = 559.83 µM) [45]. RU from Artemesia judaica L. (A. judaica L.) ethanolic extract showed apoptotic potential against NSCLC cells and arrested the cell cycle at the G2/M phase. A cytotoxic activity of A. judaica L. extract was reported against A549 cells (IC₅₀ = 14.2 μg/mL). This may be due to the presence of several extracts of different polarities [46]. RU has shown antioxidant effects on NSCLC cells by decreasing NNK-induced intracellular ROS and inhibiting the DNA damage induced by β-carotene [47]. Thus, RU may have therapeutic potential in nicotine-induced NSCLC cells by inhibiting proliferation, migration, and adhesion, and promoting apoptosis. RU also has a beneficial effect as a potential antioxidant in inhibiting NNK-induced DNA damage in NSCLC cells.

Isorhamnetin (IS) (3′-methoxy-3,4′,5,7-tetrahydroxyflavone), a flavonol compound found in the leaves of medicinal plants such as Hippophae rhamnoides L. (H. rhamnoides L.) and Ginkgo biloba L. (G. biloba L.) has a wide range of therapeutic effects against several diseases, such as cerebrovascular diseases and atherosclerosis, It has also shown anti-tumor effects against various common cancers, including LC [48]. A few in vivo studies so far have shown a high IS bioavailability. Phytic acid improves the oral absorption of flavone compounds of H. rhamnoides L, including IS [49]. The bioavailability of IS increased when it was coingested with G. biloba L. extract solid dispersions and phospholipid complexes [50].

The therapeutic effects of IS against nicotine-induced NSCLC may be related to the inhibition of α7nAChR and its downstream signaling pathways. However, whether IS has potential α7-PAM activity remains unknown. IS is structurally similar to Q, which has an OH group at C-3. IS has four OH groups at the C-3,5,7,4′ positions, with one OH group on the B and C rings and two OH groups on the A ring [27,28]. IS also has one methoxy group in the C-5′ position on the B ring [27,28]. Thus, IS may be an effective anti-NSCLC agent by binding to the molecules involved in the α7nAChR-mediated signaling pathways.

Another possible explanation for the anti-tumor activities of IS against nicotine-induced NSCLC might be due to the enzymatic de-glycosylation of IS by α-R and β-G [51], which could lead to improved efficacy of IS for the treatment of NSCLC. IS may interact with the active site of both α-R and β-G, resulting in the inhibition of signaling pathway regulation of cellular processes implicated in nicotine-induced NSCLC.

IS may have anti-proliferative and apoptotic/autophagic effects against nicotine-induced NSCLC via regulation of α7nAChR and its downstream signaling pathways. IS inhibits proliferation/colony formation ability and promotes the apoptosis/autophagy of NSCLC cells in a time and dose-dependent manner via the mitochondria-dependent apoptosis pathway [52]. Treatment with IS demonstrated significant inhibition of migration and invasion via inactivation of the oncogenic kinase signaling pathway [53].

Hyperoside (HP), also termed Q 3-O-β-D-galactopyranoside, a naturally occurring flavonol that is widely present in plants such as Polygonum aviculare, Crataegus pinnatifida, and Hypericum monogynum, exerts a wide range of anticancer effects [29,54]. The therapeutic effect of HP on nicotine-induced NSCLC is still unclear, but it may be attributed to its aglycone Q, which has a great binding affinity for human α7nAChR [29]. HP may have high affinity and potential for binding to the active site of α7nAChR. The anti-NSCLC activity of HP may depend on its having eight OH groups on the A, B, and C-rings of their structure, with two OH groups on the B ring (positions 3′ and 4′), two OH groups on the A ring (positions 5 and 7), and four OH groups in the glycosides linked to the C ring (positions 2″, 3″, 4″, and 5″) [27,29]. In vivo, low HP bioavailability may be due to its poor oral absorption [55,56].

A few studies suggest that HP treatment exhibits a range of effects against nicotine-induced NSCLC, including anti-proliferative, anti-migration, anti-invasion, anti-inflammatory, and apoptotic/autophagic activities. HP has been reported to suppress proliferation and promote apoptosis of T790M-positive NSCLC cells by increasing forkhead box protein O1 (FoxO1) expression in colon cancer-associated transcript 1 (CCAT1)-knockdown NSCLC cells [57]. HP inhibits the expression of genes associated with tumor migration and invasion in NSCLC cells by suppressing signaling pathways involving tumor metastatic genes [58]. HP decreases viability and induces apoptosis of NSCLC cells by upregulating pro-apoptotic-related gene expression through activation of apoptotic pathways [59]. HP activates apoptosis and autophagy in proliferating NSCLC cells by increasing apoptotic/autophagic-related gene expression via a range of signaling pathways associated with tumorigenesis [60]. HP regulates genes involved in apoptosis while downregulating genes involved in proliferation, migration, invasion, and inflammation of NSCLC cells via inhibiting the NF-kB signaling pathway [61]. HP inhibits proliferation, increases apoptosis, and causes arrested growth of NSCLC cells at the G1/S phase by decreasing the protein expression of cyclin-dependent kinase (CCND1) and coding sequence (CDK 4 & 6) through its interaction with the microRNA-let7a-5p [62]. HP significantly inhibited proliferation, migration, invasion, and angiogenesis; induced apoptosis; and arrested the cell cycle at the S phase in NSCLC cells, mediated through upregulating pro-apoptotic and downregulating anti-apoptotic protein levels [63]. HP reduces the inflammation of NSCLC cells by decreasing Mycoplasma pneumoniae pneumonia (MPP)-induced pro-inflammatory cytokines production through NF-κB signaling pathway inactivation [64]. HP reduces hypoxia-induced viability and proliferation of NSCLC cells, as demonstrated by upregulating the expression of heme oxygenase-1 (HO-1) through activating AMP-activated protein kinase (AMPK) [65].

Rhamnetin (RT) and rhamnazin (RZ) are methylated QDs distributed widely in fruits and vegetables that exert antibacterial, anti-inflammatory, antioxidant, and anticancer properties [27,66]. Previous evidence has demonstrated that the methoxy groups produce steric hindrance and reduce the free radical scavenging ability of flavonoids. However, the OH groups endow flavonols with high radical scavenging capability [67]. RT and RZ are structurally similar to IS but differ from other flavonols by having methoxy groups on the A and B rings [27,28]. RT (7′-O-methoxy Q) contains a methyl group at the 7′ position on the A ring, while RZ (3′,7′-dimethylquercetin) has two methyl groups at the 3 and 7′ positions on the A and B rings [27,30]. RT and RZ have an OH group at C-3, which plays a significant role in anti-tumor activity against nicotine-induced NSCLC. RT possesses four OH groups at C-5, 3, 3′, and 4′, with one OH group on the A and C rings and two OH groups on the B ring. RZ has three OH groups in its structure at the 5 position on the A ring, the 3 position on the C ring, and the 4′ position on the B ring [27,30].

RT has been shown to displace a selective α7nAChR ligand, methyllycaconitine [³H]-MLA, or an αβ2 selective ligand, [³H]-cytisine, with the lowest cytotoxicity activity (IC₅₀) of nicotine, resulting in inhibited NO and TNFα release [68]. RT also exerts α7nAChRs agonist activity in vivo, as demonstrated by reduced excitotoxicity following ethanol exposure and neuroinflammation by decreasing lipopolysaccharide (LPS)-induced TNFα and NO production [69].

RZ may have a significant role in anti-angiogenic effects in NSCLC cells, as demonstrated by a xenograft mouse model. RZ suppresses in vivo angiogenesis mediated by NSCLC cells by inhibiting vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation through anti-programmed cell death 1/programmed cell death ligand 1(PD-1/PD-L1) signaling pathway inactivation. Treatment with RZ (20 μM) and anti-CD3 antibody (1 μg/mL) exhibits cytotoxic effects on NSCLC cells, promotes T cell proliferation, and increases the production of cytotoxic mediators [70].

Quercitrin (QU), also known as Q-3-O-rhamnoside, is a Q derivative that is structurally similar to HP. QU has seven OH groups on the A, B, and C rings, with two OH groups on the A ring (positions 5 and 7), two OH groups on the B ring (positions 3′ and 4′) and three OH groups in the glycosides linked to the C ring. QU differs from QDs by having one methyl group in the glycosides linked to the C ring, which makes its anti-NSCLC ability lower than the other derivatives [27,30]. Thus, Qu would be expected to have lower levels of effectiveness as an anti-NSCLC agent than other QDs in binding to the α7nAChR-mediated signaling pathways.

A few studies have demonstrated that QU exerts anti-proliferative, anti-migration/invasion, and apoptotic effects on NSCLC cells. QU inhibits proliferation and induces apoptosis in NSCLC cells via increasing caspase-3 enzyme activity and the nucleosomal enrichment factor, and inducing the loss of mitochondrial membrane potential. QU exerts cytotoxic effects on NSCLC cells in a dose-dependent manner (50 μmol), which may be due to inducing lactate dehydrogenase (LDH) release to baseline levels of toxicity [71]. NSCLC cell migration and invasion has been shown to be reduced by QU treatment at different concentrations by inhibiting the transcript levels of Gap Junction Protein Beta 2 (GJB2) in these cells [72].

Tamarixetin (TA), also called 4′-O-methoxy Q, is a derivative that has four OH groups on C 3, 5, 7, and 3′ [30], among which the OH group at C-3 may improve the anti-tumor activity against NSCLC [67]. However, TA has a methyl group at the 4′ position on the B ring [30]. Only one study showed that TA and IS inhibit the proliferation of NSCLC cells. This inhibition was accompanied by increased apoptosis-related gene expression. Cytotoxicity analysis has indicated cytotoxic activity of TA and IS with an IC₅₀ value between 15–26 μM in NSCLC cells. This may be due to the combination of different flavonoids, which increases the cytotoxic potencies of these methylquercetins [73].