Autophagy is a type II programmed cell death that prevents tumor initiation and suppresses cancer progression in early tumorigenesis. As a key regulator of cellular metabolism during starvation, autophagy normally protects cells from stressors like nutrient deprivation. (Kimmelman and White, 2017). Stimulation of the tumor microenvironment usually triggers autophagy, such as nutrient deprivation, reactive oxygen species (ROS), hypoxia, and pathogen invasion (Su et al., 2015). Typically, autophagy passes through distinct stages including induction of autophagy, nucleation of the autophagosome, expansion, and elongation of autophagosomal membranes, closure, and fusion of autophagosomes with lysosomal membranes, and degradation and recirculation of intracapsular products (Li et al., 2020). During cancer cell survival, autophagy plays dichotomous role, exerting dynamic tumor-suppressive or tumor-promoting effects at different stages or settings (White, 2015; Levy et al., 2017). Although autophagy promotes tumorigenesis, abundant evidence indicates that the triggering of autophagy may limit tumor progression and improve response to cancer therapy (White, 2015; Amaravadi et al., 2019). In addition to the cytoprotective and cytotoxic forms of autophagy, Gewirtz DA proposed in a review published in 2014 that autophagy is actually populated by at least two additional players, a nonprotective form of autophagy and a cytostatic form of autophagy (Gewirtz, 2014). As a survival response, cytoprotective autophagy enables tumor cells to evade apoptotic signals and become resistant to chemotherapy and radiotherapy. When cytoprotective autophagy is blocked, it can enhance the sensitivity of tumor cells to chemotherapy and increase apoptosis in cancer cells (Ulasov et al., 2020; Xu et al., 2022). For this reason, multiple clinical trials are currently being conducted for treating cancer by targeting cytoprotective forms of autophagy using autophagy inhibitors (e.g., chloroquine or hydroxychloroquine) in combination with various conventional therapeutic modalities tto achieve improved efficacy (Gewirtz, 2014). Although autophagy promotes tumorigenesis, abundant evidence indicates that the triggering of autophagy may limit tumor progression and improve response to cancer therapy (White, 2015; Amaravadi et al., 2019). Cytotoxic autophagy promotes tumor cell death, either by killing its own cells or by acting as a precursor to apoptosis (Sui et al., 2013; Gewirtz, 2014). Generally, the key to distinguish between cytotoxic autophagy and cytoprotective autophagy is to observe the sensitivity of tumor cells to the therapeutic modality. Besides, Thorburn A and Gewirtz DA observed induction of another form of autophagy by radiation, which would be termed ‘‘nonprotective’’, whose inhibition is neither affect cell proliferation nor apoptosis (Bristol et al., 2013). In a particular tumor cell line, the manner of treatment may determine whether autophagy is cytoprotective or nonprotective (Gewirtz, 2016). In 2014, Gewirtz DA proposed for the first time a novel form of autophagy, namely cytostatic autophagy (Sharma et al., 2014). This study noted that combined treatment with vitamin D (or a vitamin D analogue, EB 1089) and radiation resulted in more pronounced growth inhibition in non-small cell lung cancer cells than radiation alone, as well as greater sensitivity to radiation. In addition, they identified that radiation-induced conversion of cytoprotective autophagy to cytostatic autophagy. Considering that cytostatic autophagy usually mediates cell growth inhibition, this form of autophagy may affect the effectiveness of chemotherapy and/or radiotherapy targeting tumor cell growth arrest. Unfortunately, there is no well-defined biochemical or molecular signature that distinguishes these forms from each other.

Quercetin has been demonstrated to promote and control the regulation of autophagy in different types of cancers. Treatment of Burkitt lymphoma cell lines with quercetin, LC3Ⅰ was converted to LC3Ⅱ, which is commonly considered a biomarker of autophagy, suggesting that quercetin induced a complete autophagic flux (Granato et al., 2016). The same findings were obtained in HL-60 xenograft mice, where quercetin administration induced the conversion of LC3-I to LC3-II and activated autophagy proteins, indicating that quercetin treatment triggered the autophagic process and thus anti-tumor growth (Calgarotto et al., 2018). In addition to upregulating the LC3-II/I ratio in a dose-dependent manner, a large number of double membranes were observed in quercetin-treated glioma cell lines (Bi et al., 2016). In SH-SY5Y cells, quercetin also induced autophagy by upregulating LC3II. This study also revealed that quercetin restored organelle endoplasmic reticulum homeostasis and alleviated the cytotoxic damage induced to Cu by regulating the autophagic pathway (Chakraborty et al., 2022). Moreover, the highest number of cellular autophagic vacuoles was observed in HeLa cells treated with 50 μM quercetin, but the number decreased instead at higher doses (Wang et al., 2016). In lung cancer cells, quercetin treatment also significantly increased Beclin 1 protein expression and the number of autophagic vacuoles and autophagosomes (Guo et al., 2021). Contrasting results were observed in human glioblastoma cell lines, where quercetin treatment increased the formation of autophagic lysosomal vesicles but had no effect on the expression of Beclin-1 (Kim et al., 2013). Mammalian target of rapamycin protein (mTOR) is one of the key proteins regulating the autophagic process, causing phosphorylation and inactivation of autophagy protein (ATG), thus inhibition of mTOR leads to upregulation of ATG and initiation of the autophagic process (Kim et al., 2011; Kim and Guan, 2015). In an in vivo and in vitro study, quercetin was used to treat breast cancer. This study indicated that quercetin induced cellular autophagy by inactivating the protein kinase B (Akt)-mTOR pathway, while the use of Akt-mTOR pathway inducers and autophagy inhibitors further confirmed the involvement of the Akt-mTOR pathway in quercetin-induced autophagy (Jia et al., 2018). Similarly, autophagosomes and autophagolysosomes were significantly increased in hepatocellular carcinoma (HCC) cells after quercetin treatment. By using pathway-specific inhibitors or activators, it is suggested that quercetin stimulates autophagy by inactivating the AKT/mTOR pathway and activating the MAPK pathway (Ji et al., 2019). In addition, quercetin significantly induced cellular autophagy and enhanced gemcitabine-induced cytotoxicity by inhibiting the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR axis in pancreatic cancer cells (Lan et al., 2019). In addition, quercetin may also induce autophagy in cancer cells through other mechanisms. Wu et al. found that quercetin treatment induced cellular autophagy by upregulating LC3 expression and downregulating p62 expression in a time-dependent manner. These effects were at least partially dependent on quercetin downregulation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) activation (Wu et al., 2019). In primary ovarian cancer (OC) cells, quercetin treatment activates the p-STAT3/Bcl-2 axis followed by induction of protective autophagy (Liu, Gong, et al., 2017). In the study of quercetin-induced osteosarcoma cell death, quercetin promoted the expression of autophagy-related genes through activation of NUPR1 gene activity, which subsequently triggered excessive autophagy in cancer cells (Wu et al., 2020). There are complex interactions between autophagy and apoptosis. A study confirmed that quercetin significantly enhanced tumor necrosis factor (TNF)-related apoptosis-induced ligand-mediated lung cancer cell death through activation of autophagy (Moon et al., 2015) (Figure 2).

Interestingly, the combination of quercetin with other drugs can improve the antitumor efficacy of quercetin by inhibiting or inducing autophagy. Granat et al. demonstrated that quercetin treatment induced a complete autophagic flux in the primary effusion lymphoma (PEL) cell lines. Furthermore, further accumulation of the lipidated form of the autophagy marker LC3 (LC3-II) was observed in quercetin combined with vesicular proton pump inhibitor-treated BC3, BCBL1, and BC1 cells compared to the single treatments (Granato et al., 2017). Another study demonstrated that quercetin-induced autophagy reduced its therapeutic effect on gastric cancer (GC) cells, and treatment with quercetin combined with miR-143 agonist, an inhibitor of autophagy in GC cells targeting GABARAPL1, could improve the antitumor efficacy of quercetin (Du et al., 2015). In addition, quercetin combined with soluble epoxide hydrolase inhibitor (t-AUCB) promotes cell death by inducing autophagy blockade, which may be a potential strategy for the treatment of glioblastoma (Li J. et al., 2016a).

Cellular senescence, a permanent state of cell cycle arrest due to various cancer-induced stresses, inhibits cancer by irreversibly preventing cell proliferation and is one of the protective mechanisms against cancer in addition to apoptosis (Collado et al., 2007; Calcinotto et al., 2019). Cellular stress, DNA damage, and oncogene activation are among the stimuli that cause cellular senescence (Ma et al., 2018; Jochems et al., 2021). Besides the cell cycle arrest, the morphological features that accompany cellular senescence include cellular enlargement, flattening, vacuolization, and occasionally multinucleation or increased nuclear occupancy. However, these changes are usually only observed during cellular senescence in vitro cultures, while in vivo senescent cells maintain normal morphologically determined tissue structure (Collado and Serrano, 2006; Muñoz-Espín and Serrano, 2014; Bernadotte et al., 2016). In cultured cells and/or tissues, the detection of a collection of biomarkers is used to define senescence. The histochemical assay for β-galactosidase activity is the most widely used assay for senescence. Common mediators of senescence, including p16, ARF, p53, p21, p15, and p27, are also typical biomarkers of senescence (Muñoz-Espín and Serrano, 2014). In addition, alterations in Lamin B1 levels are a common feature of many types of senescence (Lukášová et al., 2018; Radspieler et al., 2019). Generally, senescence is considered as an effective anti-tumor mechanism through which cancer cells proliferate and inhibit malignant progression. Furthermore, senescence is one of the physiological tumor suppressor mechanisms that limit the progression of tumors from benign tumor lesions to malignant ones. As a consequence of these effects, it has cancer suppressive potential, while senescence-associated secretory phenotype (SASP) plays an important role in the pathophysiological role of senescent cells (Wyld et al., 2020; Özsoy Gökbilen et al., 2022). However, the signaling pathway of senescence is also a key effector of radiotherapy and chemotherapy injury, which may lead to reduced recovery in patients receiving anticancer therapy and may result in cancer recurrence. On the other hand, growing evidence indicates that senescent cells may induce proliferative pathology in cancer, moreover, SASP factors may also trigger epithelial-mesenchymal transition (EMT) in premalignant epithelial cells (Liu and Hornsby, 2007; Kuilman et al., 2008). Therefore, the use of senolytic agents to remove senescent cells may play a key role in preventing cancer recurrence.

It was found that quercetin can induce senescence in cancer cells. In quercetin-treated T24 bladder cancer cells, an increase in the percentage of nuclei during cellular senescence was observed by nuclear morphometric analysis (NMA) (Adami et al., 2021). Furthermore, the immunoreactivity of Lamin B1, p16, and cyclin B1, markers of cellular senescence, was also increased in Colo-320 and Colo-741 cells after treatment with quercetin (Özsoy et al., 2020). Pan et al. found that the mechanism by which quercetin promotes cellular senescence may be via inhibition of the Ras/MAPK/ERK signaling pathway in a dose-dependent manner (Pan et al., 2015). In another study, treating C6 and U87 cells with quercetin for 4 consecutive days elevated levels of cellular senescence markers, furthermore senescence-associated cell morphological changes such as flattening, increased particle size, and cell enlargement could be observed. The researchers also found that histone deacetylase (HDAC) played a key role in quercetin-induced senescence. HDAC inhibitors significantly enhanced quercetin-induced senescence in human and rat glioma cell lines (Vargas et al., 2014). Quercetin triggers cellular senescence probably by increasing the expression of tumor suppressor gene p53 and cell cycle protein-dependent kinase (CDK) and cyclin B1 inhibitors p21, p27, thus inducing cell cycle arrest in G1 and G2/M phases (Tang et al., 2017; Özsoy Gökbilen et al., 2022). A study in 2013 showed that quercetin treatment for 18 h increased Hela cell density in the G2/M phase of the cell cycle, reflecting cell cycle arrest at this stage (Bishayee et al., 2013). Moreover, Liu et al. confirmed the same finding in different cell lines. After quercetin treatment for 24 h, the number of U251 glioblastoma cells in the sub-G2/M phase increased, indicating that quercetin induced G2/M phase arrest and thus inhibited U251 cell proliferation (Liu, Tang, et al., 2017). Cantanzaro et al. designed a study to investigate the factors by which quercetin affects the G1/S and G2/M cell cycles. The results showed that quercetin treatment on SKOV3 and U2OSPt cells for 48 h significantly altered the cell cycle distribution, possibly affecting the G1/S and G2/M phases of the cell cycle by decreasing the levels of cyclin D1 and cyclin B1 (Catanzaro et al., 2015).

Quercetin is usually not as powerful as targeted senolytics agents, but quercetin in combination with other senolytic agents can be more effective in removing senescent cells. Zhu Y et al. first reported a senolytic cocktail (Dasatinib combined with quercetin) that effectively killed senescent cells with the help of small interfering RNA (Zhu et al., 2015). Subsequently, in two open-label Phase I pilot studies, researchers demonstrated for the first time that senolytic agent (Dasatinib combined with quercetin) significantly reduces human senescent cell burden and provided preliminary evidence that senolytic agents may alleviate physical dysfunction in patients with idiopathic pulmonary fibrosis (IPF) (Hickson et al., 2019; Justice et al., 2019). Notably, some chemotherapeutic agents that exert their antitumor effects via the induction of cancer cell senescence also trigger cellular senescence in normal cells, which drives the development of a malignant phenotype in residual living tumor cells (Demaria et al., 2017; Bruni et al., 2019). Quercetin has been demonstrated to reduce chemotherapeutic drug-induced aging in combination with anticancer drugs. Recent studies have established that quercetin pre-treatment can prevent doxorubicin-induced senescence in normal cells by reducing the number of senescent cells and the production of SASP factors (Bientinesi et al., 2022). Furthermore, quercetin pre-treatment may also protect normal cells from doxorubicin treatment-induced ROS damage, by increasing cellular antioxidant defense. In another study, a quercetin derivative (quercetin-3-O-β-D-glucuronide) also showed a good inhibitory effect on cellular senescence in doxorubicin-treated HDFs and HUVECs cells (Yang et al., 2014). Considering that cancer cells may use senescence as an escape strategy from cancer treatment, the use of quercetin may selectively remove spontaneous cancer cells previously induced by chemotherapy and/or radiotherapy. Therefore, the use of some phytochemicals as senolytic agents or protectors, such as quercetin, is probably useful for overcoming tumor resistance (Figure 3).

Mitotic catastrophe is another critical non-apoptotic mechanism of cancer cells, defined by the Nomenclature Committee on Cell Death (NCCD) in 2012, which usually occurs after massive damage to DNA following ROS generation (Galluzzi et al., 2012). Cells undergoing mitotic catastrophe are often in partnership with other mechanisms of cell death, such as autophagy, senescence, or, necroptosis (Eom et al., 2005; Zhao W. et al., 2022b; Egorshina et al., 2022). Recent studies have found that the therapeutic efficiency of anticancer modalities such as radiotherapy and chemotherapy can be reinforced by stimulating mitotic catastrophe, which is also a promising way to overcome multidrug resistance (Pérès et al., 2015; Bai et al., 2017). In addition, ionizing radiation (IR) can also trigger immune cell mitotic catastrophe (Adjemian et al., 2020). Hence, the stimulation of mitotic catastrophe constitutes a new direction for tumor therapy. However, very few studies have evaluated the effects of quercetin on mitotic mutations, and the available studies have only found that quercetin induced mitotic mutations, but precisely little has been done to characterize exactly what signals are involved. Jackson et al. characterized the effect of different concentrations of quercetin on mitotic mutagenesis in Hepa1c1c7 cells. The results demonstrated that aberrant mitotic images were observed at concentrations as low as 0.01 μM quercetin, showing disproportionate DNA segregation (Jackson et al., 2016). Sufficient evidence documented that such mitotic abnormalities may eventually lead to mitotic catastrophes (Zhu et al., 2005). Thus, it appears that utilizing lower doses of quercetin to trigger mitotic catastrophes would significantly limit the side effects of the administration of large doses of quercetin anti-tumor. In another study, to investigate whether quercetin can induce growth inhibition in A549 cells by altering the cell cycle, image cytometric analysis of cellular DNA content was used to assess the effect of quercetin on cell cycle distribution. It was found that quercetin treatment dose-dependently resulted in a decrease in cell cycle distribution in G₀/G₁-phase accompanied by an increase in cell cycle distribution in G₂/M-phase in A549 cells (Kobayashi et al., 2008). Moreover, the researchers attribute the failure of cytokinesis to quercetin-induced mitotic catastrophe, with monopolar spindle formation caused by perturbation of mitotic microtubules as a possible mechanism. However, the mechanism of quercetin in regulating mitotic catastrophe needs further elucidation (Figure 3).

Ferroptosis, a novel mode of cell death first introduced in 2012, depends on intracellular iron and differ from apoptosis, necroptosis, and autophagy in morphology and biochemistry (Dixon et al., 2012; Lei et al., 2022). Ferroptosis is mainly characterized by iron ion accumulation and ROS-induced lipid peroxidation, which morphologically induces marked mitochondrial contraction, increased membrane density, and reduction or disappearance of mitochondrial cristae (Xie et al., 2016; Yang and Stockwell, 2016). The important role of ferroptosis in oxidative stress, iron metabolism, inflammation, and amino acid metabolism has been convincingly established (Galaris et al., 2019; Sun et al., 2020; Yang J. et al., 2022b). Correspondingly, ferroptosis involves multiple physiological and pathological processes, including hematological disorders, ischemia-reperfusion injury, renal injury, and particularly tumor inhibition (Mou et al., 2019; Chen et al., 2021; Li et al., 2021; Zhao et al., 2021).

Recently, it has been found that quercetin exerts anti-tumor effects via triggering ferroptosis to induce cancer cell death. The activation of ferroptosis involves multiple signaling pathways. Recent research has identified a new ferroptosis-inducing pathway stimulated by autophagy, namely autophagy-dependent ferroptosis, which is selective autophagy that contributes to ferroptosis (Pierzynowska et al., 2021). During autophagy, the macromolecules in need of degradation are engulfed by phagophores, followed by acid hydrolase digestion of their contents by the lysosome. Transcription factor EB (TFEB) is the master gene for lysosomal biogenesis and autophagy (Settembre et al., 2011; Medina et al., 2015). These findings enlighten us that lysosomal storage diseases (LSD) and related molecular pathogenesis may involve the regulation of ferroptosis. Wang et al. found that the induction of cell death by quercetin could be reversed by lysosomal inhibitors and knockdown of the TFEB, which indicated the involvement of lysosomes in quercetin-induced cell death (Wang Z. X. et al., 2021b). In addition, quercetin promotes ferritin degradation, free iron release, and lipid peroxidation, which was induced by promoting nuclear TFEB and transcriptional activation of lysosomal genes that induce lysosomal activation and inducing ROS production. The synergistic effect of both together leads to iron death. Interestingly, another research also demonstrated that TFEB-mediated lysosomal activation plays an important role in quercetin-induced ferroptosis (An and Hu, 2022). In breast cancer cells, quercetin treatment induced the onset of ferroptosis by promoting TFEB expression and nuclear transcription. Further mechanistic studies showed that the degradation of ferritin and release of ferric ions were regulated by the lysosome-related gene LAMP-1, which was up-regulated due to the high expression of TFEB in the nucleus. Notably, quercetin exhibits a therapeutic effect in several non-tumor disease models due to its significant antioxidant activity characterized by reduced malondialdehyde (MDA) and lipid ROS levels and increased glutathione (GSH) levels, which contradicts the induction of ferroptosis in tumor cells (Wang Y. et al., 2021a; Jiang et al., 2022). ROS is a double-edged sword, and the heterogeneity of tumor cells and non-tumor cells leads to their different responses to ROS. Therefore, quercetin may exhibit opposite abilities in regulating ROS in different cells. Thus, the mechanisms and signaling pathways of quercetin regulation of ferroptosis for cancer treatment still need to be further elucidated (Figure 4).

As another important mechanism of cancer cell death, necroptosis was initially found to be an alternative to apoptosis following the involvement of death domain receptors (Degterev et al., 2005). Triggering necrosis in cancer cells is a promising way to avoid the failure of cancer chemotherapy due to apoptosis resistance by bypassing the apoptotic pathway to induce cancer cell death. As such, the molecular mechanisms of necroptosis have been well investigated, which depends critically on receptor-interacting serine-threonine kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL), regardless of the upstream trigger (Hu et al., 2022; Yan et al., 2022). Currently, only limited experiments have shown the induction of necroptosis in cancer cells by quercetin. A study was conducted to observe the ultrastructural changes of quercetin on giant cell tumor of bone (GCTB) cells. The researchers demonstrated that, in addition to autophagy, quercetin treatment affected all histological components of necroptosis and secondary necroptosis by increasing the expression of RIPK1 (Estrada-Villaseñor et al., 2021). Another study reported that quercetin treatment of MCF-7 breast cancer cells depended on multiple cell death pathways, which mainly involve necroptosis (Khorsandi et al., 2017). This study indicated that quercetin induced necroptosis mainly through increasing the expression of RIPK1 and RIPK3. However, other studies indicated that quercetin treatment inhibits M1 macrophage/microglia polarization after spinal cord injury by inhibiting signal transducer and activator of transcription-1 (STAT1) and nuclear factor kappa-B (NF-κB) pathways, which ultimately results in partial alleviation of necrosis of Oligodendrocytes (Fan et al., 2019). The contradictory findings suggest that quercetin may possess the ability to specifically identify and kill tumor cells, which results in opposed effects in tumor cells and non-tumor cells. However, numerous types of research are still needed to confirm this conjecture (Figure 4).

Pyroptosis, programmed cell death in the form of inflammation, is mediated by the gasdermin family (GSDMs) (Kovacs and Miao, 2017). In the canonical pathway of pyroptosis, certain inflammasomes drive cysteinyl aspartate specific proteinase-1 (Caspase-1) activation, leading to cleavage of gasdermin D (GSDMD) and activation of the inactive cytokines like interleukin-18 (IL-18) and interleukin-1beta (IL-1β), ultimately triggering pyroptosis (He et al., 2015; Schneider et al., 2017). Recent evidence suggests that pyroptosis induces a strong inflammatory response and shows a strong tumor regression effect (Fang et al., 2020). Currently, quercetin has not been reported to possess anti-tumor effects through the regulation of pyroptosis. However, it has been investigated that the specific role of quercetin in breast cancer-related depression (BCRD), in which the inhibition of pyroptosis and promotion of immune response are the main mechanisms for effectively mitigating the progression of BCRD (Zhu et al., 2022). In this study, quercetin partially reversed the pyroptosis on LPS-cultured 4T1 cells in vitro, as evidenced markedly by upregulating the card structural domain, NLR family pyrin structural domain (NLRP3), and Caspase-1. In addition, quercetin also promoted an anti-tumor immune response in xenograft mice. Quercetin treatment significantly upregulated the levels of interferon-gamma (IFN-γ), interleukin-10 (IL-10), and interleukin-2 (IL-2) in BCRD Mice. However, the modulation pathways of pyroptosis, especially the immunological effects, remain to be further elucidated after the treatment of cancer with quercetin.