Molecular Mechanisms Underlying the Anti-Cancer Effects of Tangeretin, a Phytochemical from Citrus Extracts

Abstract

Cancer is one of the diseases with high incidence and mortality rates. As a result, many studies have led to the development of therapeutic agents such as immune checkpoint inhibitors. However, the research on cancer treatment is still essential because of problems including drug resistance, genetic variation among patients, and drug toxicity. To address these problems, several candidate chemicals, including phytochemicals, have been studied for cancer treatment. Phytochemicals exhibit a variety of biochemical and physiological functions in the body and have been studied as having lower toxicity than synthetic chemicals. These properties have led to research into their potential as anti-cancer agents as well as their therapeutic applications for a variety of other diseases. The structural diversity of phytochemicals leads to considerable variations in their mechanisms of action. Therefore, it is important to understand the mechanism of action of each phytochemical when studying phytochemicals. Tangeretin (TAN) is mainly extracted from citrus fruits and is characterized by the presence of a methyl group. The presence of a methyl group in TAN is thought to enhance its intracellular uptake and confer greater resistance to degradation compared with other phytochemicals. Previous studies on the use of TAN in cancer treatment have mainly focused on its ability to induce oxidative stress as the primary anti-cancer mechanism. In addition, some studies suggest that TAN also exerts various other anti-cancer effects, such as upregulating tumor suppressor proteins, inducing apoptosis, and reducing the proportion of cancer stem cells (CSCs). This review highlights the anti-cancer effects of TAN in different cancers and aims to provide data to support future research.

INTRODUCTION

Cancer is the second leading cause of death worldwide, after cardiovascular disease. As of 2022, lung cancer is the leading cause of cancer-related deaths, followed by colorectal, breast, stomach, and esophageal cancers (Bray et al., 2024; Jemal et al., 2010; Siegel et al., 2019). Although various research and development efforts are conducted to treat cancer, cancer treatment remains challenging due to various problems, including immune evasion, autocrine action, and drug resistance of cancer cells. Immune evasion of cancer cells is mainly activated by immune checkpoint proteins (ICPs), such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1), and has been targeted in various studies for cancer treatment (Wang et al., 2024). In addition, cancer cells generate autocrine effects, in which cancer cells produce and recognize cytokines and growth factors on their own. Autocrine effects are also the cause of drug resistance in various cancers and may be an important target for cancer therapy (Jia et al., 2017). Drug resistance is one of the major obstacles to effective cancer treatment and is often associated with the expression of ATP-dependent transporters and antioxidant mechanisms (Kaur et al., 2020). ATP-binding cassette (ABC) transport anti-cancer drugs such as cisplatin (DDP) and paclitaxel (PTX) out of cancer cells, reducing drug accumulation within cancer cells and reducing treatment effectiveness (Bergonzini et al., 2024; Sobczak et al., 2022). Antioxidant proteins suppress the oxidative stress increased by anti-cancer drugs in cancer cells (Sayin et al., 2014). Although several chemotherapeutic agents have been developed to overcome drug resistance, the varied responses of patients to the drugs and the relatively high toxicity of currently available agents emphasize the need for research and development of new therapeutic candidates (Qi et al., 2019; Romani, 2022).

Phytochemicals have been reported to be less toxic than synthetic compounds. As a result, phytochemicals have been widely tested for use in cardiovascular disease and cancer (Bhutta et al., 2024; Malik et al., 2022). The phytochemicals are classified into various types based on their structure, such as alkaloids, organosulfur compounds, phenols, and carotenoids (Cizmarova et al., 2023; Younas et al., 2018). Flavonoids have been the topic of many research studies due to their biological activities, including anti-inflammatory and anti-cancer effects (Hazafa et al., 2020; Maleki et al., 2019). They are classified into various types, such as flavones, flavonols, flavanones, isoflavonoids, anthocyanins, and polymethoxylated flavonoids (Table 1). In addition, several studies have reported their potential protective effects on cardiovascular health and visual function (Dias et al., 2021).

Table 1.

Classification of flavonoids based on structural features and their representative biological activities

Flavonoid type	Structural features	Biological activities	References
Flavones	Flavonoid backbone witd various oxygen-containing groups at different positions	Anti-cancer, Anti-inflammatory
Flavonols	Hydroxyl group (-OH) at C3 position	Antioxidant (free radical scavenging), Cardiovascular protection
Flavanones	Absence of a double bond between C2 and C3	Anti-inflammatory, Antioxidant, Antibacterial, Antiviral	Hazafa et al., 2020; Maleki et al., 2019
Isoflavonoids	Benzene ring attached to the C3 position of the flavonoid structure	Phytoestrogenic activity, Bone health support
Anthocyanins	Flavonoid structure coupled with a sugar moiety	Potent antioxidant properties, Cardiovascular and visual function protection

Tangeretin (TAN), a polymethoxylated flavone (PMF) (Fig. 1), is mainly found in the peel of citrus fruits such as tangerines, oranges, and mandarins. TAN plays an important role in strengthening the cell walls of plants and enhancing their defense mechanisms (Raza et al., 2020). Methoxylated flavonoids are characterized by the presence of a methyl group (-CH3) in the chemical structure of flavones, which has been shown to increase their biological activity and their physiological and pharmacological properties (Walle, 2009). Previous studies have shown that TAN induces apoptosis and regulates autophagy in cancer cells. TAN also induces cell cycle arrest in cancer cells via the regulation of the expression of cyclin family proteins and retinoblastoma (Rb) proteins, which play important roles in the regulation of the cell cycle. TAN inhibits angiogenesis through the inhibition of the expression of proteins such as vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) (Boye et al., 2021).

Fig. 1.

Structure of Tangeretin.

PMFs contain a variety of bioactive compounds, including nobiletin (NOB) and sinensetin (SIN), in addition to tangeretin (TAN). A previous study compared the anti-cancer effects of TAN, NOB, and SIN in prostate cancer cells and demonstrated that TAN induced the greatest reduction in cell viability at equivalent concentrations. These findings suggest that TAN may exert superior anti-cancer efficacy compared with NOB and SIN (Chen et al., 2022). In addition, previous studies have shown that TAN’s oral bioavailability is around 27%, whereas the oral bioavailability of quercetin (QUE) and curcumin (CUR) is less than 1%. Furthermore, pharmacokinetic analyses have shown that TAN can be detected in the systemic circulation in its unmetabolised form, while QUE and CUR are undetectable in plasma. These findings provide evidence of the superior in vivo stability and bioavailability of TAN compared with other PMFs (Hung et al., 2018; Manach et al., 1997; Shen et al., 2016). Overall, studies on the anticancer effects and pharmacokinetic advantages of TAN compared with other PMFs suggest that TAN is a more promising candidate for development as an anticancer agent. Therefore, this review aims to examine the biochemical and physiological activities of TAN in cancer cells and provide insights that may facilitate future research.

ANTI-CANCER EFFECTS OF TAN IN VITRO

Lung cancer

In 2022, lung cancer was the most frequently diagnosed cancer globally and was the primary cause of cancer-related deaths (Inoue, 2024). Lung cancer is generally classified into two primary cancer types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC, which grows and spreads slowly compared to SCLC, is more common, with subtypes such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. SCLC tends to grow and spread much faster than NSCLC. Due to this aggressive characteristic, SCLC is often diagnosed at an advanced metastatic stage (Li et al., 2024a; Taniguchi et al., 2024).

Excessive accumulation of reactive oxygen species induces oxidative stress, which can lead to cell death and inhibit cancer cell proliferation (Nakamura and Takada, 2021). To counteract this, cancer cells often activate the nuclear factor erythroid 2 related factor 2 (Nrf2) signaling pathway as a protective mechanism against oxidative damage (Zimta et al., 2019). TAN has been reported to exert anti-cancer effects in NSCLC A549 cells by suppressing the expression of Nrf2, a key regulator of antioxidant defense (Xie et al., 2022). Furthermore, Nrf2 upregulates the expression of P-glycoprotein (P-gp), a key protein involved in multidrug resistance. P-gp uses ATP to export anti-cancer drugs and other toxic substances from cells (Callaghan et al., 2014; Sadeghi et al., 2018). In this study, treatment with TAN resulted in the antioxidant activity and decreased expression of P-gp through the inhibition of NF-kB protein expression. These results show that TAN may inhibit antioxidant activity in lung cancer, suppress cancer cell growth, induce apoptosis, and reduce drug resistance (Fig. 2A).

Fig. 2.

Anti-cancer effects of tangeretin in lung cancer cells. (A) Schematic illustration showing that tangeretin (TAN) inhibits the nuclear factor erythroid 2-related factor 2 (Nrf 2) signaling pathway in lung cancer cells. Suppression of Nrf 2 signaling reduces antioxidant activity and weakens cytoprotective responses, thereby decreasing drug resistance associated with Nrf2 pathway activation. (B) Diagram depicting the inhibitory effects of TAN on inflammatory and pro-survival signaling pathways. TAN reduces interleukin (IL)-1β-induced activation of the c-Jun N -terminal kinase (JNK) and Protein Kinase B (Akt) pathways and regulates cyclooxygenase- 2 (COX- 2) expression through inhibition of the nuclear factor kappa- light- chain- enhancer of activated B cells (NF- κB) pathway in lung canc er. Abbreviations: Tangeretin (TAN), Nuclear factor erythroid 2- related factor 2 (Nrf 2), Interleukin- 1 beta (IL- 1β), c- Jun N- terminal kinase (JNK), Protein kinase B (Akt), Cyclooxygenase-2 (COX-2), Nuclear factor kappa B (NF-κB).

The inflammatory cytokine interleukin IL-1β, a member of the IL-1 family, induces the production of other cytokines and results in angiogenesis, promoting epithelial-mesenchymal transition (EMT), growth, and invasion of cancer cells, thereby accelerating lung cancer metastasis (Tulotta et al., 2021; Zhang and Veeramachaneni, 2022). Cyclooxygenase-2 (COX-2), which can be activated by IL-1β-induced signaling, is primarily overexpressed in lung cancer. The production of prostaglandin E2 (PGE2) is regulated by COX-2 and is critical in promoting tumor growth and suppressing tumor immunity. PGE2 activates cancer cell growth and migration by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and c-Jun N-terminal Kinase (JNK) pathway (Kitanaka et al., 2017; Yang et al., 2020). TAN downregulated AKT and JNK protein expression by inhibiting COX-2 expression in lung cancer cells. These results suggest that TAN may inhibit oncogenic signaling pathways, such as the AKT and JNK pathways, by inhibiting IL-1β-induced COX-2 expression in lung cancer cells. Furthermore, TAN inhibits the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) induced by IL-1β signaling in lung cancer cells (Chen et al., 2007). The NF-kB pathway is hyperactivated in a variety of cancer cells and performs a variety of functions, including promoting proliferation and migration (Ebrahimi et al., 2024). The TAN-induced blockade of NF-κB translocation into the nucleus implies that TAN may inhibit additional physiological and biochemical activities in lung cancer cells (Fig. 2B).

Colorectal cancer

Colorectal cancer (CRC) has the third-highest incidence rate after lung and breast cancer, and the second-highest mortality rate after lung cancer (Inoue, 2024). RC primarily occurs in older adults and is caused by the formation of polyps in the colon and is asymptomatic in the early stages. Consequently, early detection fails for many patients who are diagnosed after the cancer has advanced (Sawicki et al., 2021).

The cell cycle consists of the G0 (resting), G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (division) phases, each of which plays an essential role in cell growth, DNA replication, and division, and each phase is tightly regulated through specific regulatory proteins, such as CDK family proteins and cyclin family proteins (Sun et al., 2021; Wiecek et al., 2023). Cyclin-dependent kinase 2 (CDK2) is a key protein that binds to cyclin E to regulate the transition from G1 phase to S phase of the cell cycle (Zhang et al., 2022) and is regulated by the tumor suppressor protein p53, which is activated by various external stresses, including anti-cancer drugs (Rastogi et al., 2015). The downstream target of p53, p21, plays an important role in blocking the G1/S transition and arresting the cell cycle by inhibiting the activity of the CDK2-cyclin E complex (Liu et al., 2018). In an earlier study, TAN significantly reduced the viability of colon cancer cells, suggesting that it may affect the survival, proliferation, or cell death in these cells. G1 phase arrest was observed in the TAN-treated group, accompanied by decreased activity of CDK2, cyclin D1, and cyclin E. In addition, the TAN-treated group showed increased levels of p53 and p21 (Pan et al., 2002). The up-regulation of p53 and p21 in TAN-treated cells suggests that TAN may restore the tumor suppressor protein p53, which induces cell cycle arrest by inhibiting CDK2 and apoptosis (Fig. 3A).

Fig. 3.

Anti-cancer effects of tangeretin in colon cancer cells. (A) Schematic representation of the cell cycle– regulatory effects of tangeretin (TAN) in colon cancer cells. TAN restores and stabilizes the tumor suppressor protein p53, leading to suppression of cyclin- dependent kinase 2 (CDK 2) activity and inhibition of CDK 2-mediated G1 phase progression. Through p53 reactivation and cell cycle control, TAN contributes to growth arrest and reduced proliferative capacity of colon cancer cells. (B) Mechanistic illustration of the combined effects of TAN and 5- fluorouracil (5-FU) in colon cancer cells. Co- treatment with TAN and 5-FU decreases antioxidant activity, enhances oxidative stress, and promotes activation of the intrinsic (mitochondria- mediated) apoptotic pathway, resulting in increased apoptotic cell death compared with single treatment. Abbreviations: Tangeretin (TAN), Cyclin- dependent kinase 2 (CDK 2), 5- Fluorouracil (5-FU), G1 phase (Gap 1 phase).

Similarly, TAN has also been reported to act synergistically with chemotherapeutic agents. A recent study has revealed that TAN, when co-treated with 5-fluorouracil (5-FU), resulted in an increase in intracellular ROS levels due to the suppression of oxidative stress-suppressing proteins. Additionally, the TAN and 5-FU co-treatment decreased mitochondrial membrane potential (MMP) compared with TAN or 5-FU alone. The p21 level decreased in the single-treatment groups but increased in the TAN and 5-FU co-treatment group. In addition, the TAN and 5-FU co-treatment decreased the expression of thioredoxin (TRX), superoxide dismutase 1 (SOD-1), SOD-2, and glucocorticoid receptor (GR) proteins that are important for antioxidant activity (Hong and Park, 2021; Mi et al., 2021). These results indicate that the TAN and 5-FU co-treatment may modulate antioxidant proteins in CRC, leading to oxidative stress. TAN and 5-FU co-treatment reduced the excision repair cross-complementation group 1 (ERCC-1) protein, which recovers damaged DNA. In addition, the co-treatment also increased the number of apoptotic cells in CRC. Apoptosis-related markers such as caspase 3, caspase 9, and Bcl-2-associated protein x (Bax) were up-regulated in the co-treatment group compared with the single treatment groups (Dey et al., 2020) This study suggests that the TAN and 5-FU co-treatment increases oxidative stress, apoptosis and inhibits drug resistance by down-regulation of antioxidant proteins in CRC. In conclusion, these studies suggest that TAN may exert a synergistic effect when used in combination with other chemotherapeutic agents (Fig. 3B).

Gastric cancer

Gastric cancer is a growth of cells that starts in the lining of the stomach (Sitarz et al., 2018). According to approximations provided by the International Agency for Research on Cancer (IARC), gastric cancer was ranked fifth in the world in terms of new cases in 2022 (Inoue, 2024). The primary cause of gastric cancer is believed to be Helicobacter pylori infection. However, various other factors, including dietary and genetic factors, also contribute to the progression of the cancer (Guan et al., 2023; Lordick et al., 2022).

The extracellular apoptotic pathway can be activated by upregulation of the Fas Cluster of Differentiation 95 (CD95) and FasL (Fas ligand), which leads to Fas-associated death domain (FADD) recruitment and activation of caspase-8. The decrease of mitochondrial membrane potential (MMP) caused by various factors such as external stress is associated with the up-regulation of the BH3 interacting domain death agonist (Bid) and Bax proteins and the release of cytochrome C from mitochondria (Carneiro and El-Deiry, 2020; Chen et al., 2017; Wang et al., 2019). TAN activates the extracellular apoptotic pathway of human gastric cancer AGS cells by upregulating Fas and FasL, leading to FADD recruitment and caspase-8 activation. TAN also reduces MMP and releases cytochrome C into the cytoplasm via the up-regulation of Bid and Bax proteins. Furthermore, TAN increases the expression of p53 and p21 proteins in gastric cancer cell lines (Dong et al., 2014). This suggests that TAN may restore tumor suppressor protein function in gastric cancer and that it may serve as a promising candidate for therapeutic strategies targeting tumor suppressor pathways (Fig. 4A).

Fig. 4.

Anti-cancer effects of tangeretin in gastric cancer cells. (A) Schematic diagram illustrating the pro-apoptotic effects of tangeretin (TAN) in gastric cancer cells. TAN induces intrinsic (mitochondria- mediated) apoptosis through the release of cytochrome c from mitochondria into the cytosol, which promotes downstream caspase activation. In parallel, TAN also triggers extrinsic apoptosis by upregulating cell death receptors on the cell surface, thereby enhancing death receptor–mediated apoptotic signaling pathways and leading to increased apoptotic cell death. (B) Diagram showing the anti- metastatic effects of TAN in gastric cancer cells. TAN inhibits radiation therapy– induced epithelial– mesenchymal transition (EMT), a process associated with increased migration, invasion, and therapeutic resistance, thereby contributing to suppression of aggressive tumor phenotypes following radiation exposure. Abbreviations: Tangeretin (TAN), Cytochrome c (Cyt c), Epithelial–mesenchymal transition (EMT).

Radiation therapy (RT) elicits various anti-cancer effects, including DNA double-strand breaks and the generation of ROS (Taniguchi et al., 2024). It is widely utilized as monotherapy or in combination with chemotherapeutic agents (Jung et al., 2019; Li et al., 2024b). A recent study reported that the combination of RT with TAN exerts a synergistic anti-cancer effect. RT alone increases epithelial-mesenchymal transition (EMT) in gastric cancer cells by upregulating associated proteins, such as N-cadherin. However, the addition of TAN to RT was found to attenuate RT-induced EMT (Zhang et al., 2015). These findings suggest that the combined application of RT and TAN may effectively inhibit EMT and attenuate the growth of gastric cancer cells (Fig. 4B).

Breast cancer

Breast cancer, the leading cause of mortality among women in 2022 (Bray et al., 2024), can occur due to various factors, including exposure to carcinogens, alcohol use, smoking, and genetic predisposition (Bouras et al., 2023). Breast cancer can be categorized into hormone receptor-positive breast cancer, human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and triple-negative breast cancer (TNBC). TNBC does not express hormone receptors or HER2, is believed to progress rapidly, and has a poor prognosis (Zubair et al., 2020). TAN promotes apoptosis in breast cancer cells by upregulating pro-apoptotic proteins, including Bax, caspase-3, caspase-9, and caspase-8, while downregulating the anti-apoptotic protein Bcl-2. These observations indicate that TAN induces apoptosis in breast cancer cell lines in a manner analogous to its effects on lung, colon, and gastric cancer cells. Moreover, TAN has been reported to cause a G2 phase arrest in the cell cycle by modulating cyclin B1 and D (Fan et al., 2019). These findings suggest that TAN induces apoptosis in breast cancer cells and inhibits the proliferation of these cells (Fig. 5A).

Fig. 5.

Anti-cancer effects of tangeretin in breast cancer cells. (A) Schematic diagram illustrating the pro-apoptotic effects of tangeretin (TAN) in gastric cancer cells. TAN induces intrinsic (mitochondria- mediated) apoptosis through the release of cytochrome c from mitochondria into the cytosol, which promotes downstream caspase activation. In parallel, TAN also triggers extrinsic apoptosis by upregulating cell death receptors on the cell surface, thereby enhancing death receptor– mediated apoptotic signaling pathways and leading to increased apoptotic cell death. (B) Diagram showing the anti- metastatic effects of TAN in gastric cancer cells. TAN inhibits radiation therapy– induced epithelial– mesenchymal transition (EMT), a process associated with increased migration, invasion, and therapeutic resistance, thereby contributing to suppression of aggressive tumor phenotypes following radiation exposure. Abbreviations: Tangeretin (TAN), Cytochrome c (Cyt c), Epithelial– mesenchymal transition (EMT).

Of the many transcription factors involved in the regulation of breast cancer cell function, the signal transducer and activator of transcription 3 (STAT3) is phosphorylated in the cytoplasm in response to stimulation by several cytokine receptors. When STAT3 is activated, it forms a homodimer and translocates to the nucleus, where it functions as a transcription factor that promotes cancer progression, cell proliferation, and metastasis (Ma et al., 2020). CSCs expressing CD44⁺/CD24^- (Chu et al., 2024) play an important role in tumor initiation, progression, invasion, and treatment resistance. STAT3 homodimers regulate the expression of SRY-box transcription factor 2 (SOX 2), a key factor involved in the maintenance and proliferation of CSCs (Martinez-Cruzado et al., 2016). A previous study investigated the anti-cancer effects of TAN in breast cancer, with a particular focus on the regulation of the STAT3 signaling pathway. TAN has been shown to inhibit STAT3 signaling in TNBC, resulting in decreased cell proliferation and migration and increased apoptosis. Furthermore, TAN reduced the population of CD44 ⁺/CD24^- cells while inhibiting the growth and migration ability of breast cancer cells and downregulated the SOX2 protein by inhibiting the expression of STAT3 (Ko et al., 2020). This suggests that TAN may inhibit the differentiation and growth of cancer stem cells (CSCs), as well as suppress the growth and various physiological activities of cancer cells as shown in Fig. 5B and Table 2.

Table 2.

Anti-cancer effects of TAN in vitro

Cancer cell type	Major mechanisms	Key molecular targets/pathways
Lung cancer	Induction of oxidative stress, apoptosis, inhibition of migration, and drug resistance	↓ Nrf2 ↓ NF-κB ↓ P-gp ↓ IL-1β–induced COX-2 ↓ AKT/JNK signaling
Colorectal cancer	Cell-cycle arrest, apoptosis, chemosensitization	G1 arrest ↓ CDK2, cyclin D1/E ↑ p53, ↑ p21 ↑ ROS ↓ ERCC-1 ↑ caspase-3/9, Bax
Gastric cancer	Activation of extrinsic and intrinsic apoptosis, radiosensitization	Cytochrome c release ↑ Fas/FasL, FADD, caspase-8 ↓ MMP ↑ Bax/Bid ↑ p53/p21 ↓ EMT markers
Breast cancer	Apoptosis, cell-cycle arrest, CSC suppression, inhibition of migration	G2/M arrest ↑ Bax, caspase-3/8/9 ↓ Bcl-2 ↓ STAT3/SOX2 ↓ CD44⁺/CD24⁻ population

ANTI- CANCER EFFECTS OF TAN IN VIVO

Esophageal squamous cancer

Anti-cancer effects of TAN were validated not only in vitro but also in an in vivo tumor model. In a xenograft model established using esophageal squamous cell carcinoma cells in mice, administration of TAN at 20 mg/kg significantly reduced tumor weight compared with the control group. Furthermore, analysis of tumor tissues revealed that the expression levels of the proliferation marker Proliferating Cell Nuclear Antigen (PCNA) and the migration-associated protein Matrix Metalloproteinase-9 (MMP-9) were decreased in the TAN-treated group. These findings indicate that TAN may suppress tumor progression by not only reducing tumor mass but also inhibiting cancer cell proliferation and metastatic potential through the downregulation of PCNA and MMP-9.

Breast cancer

Previous in vivo study using a breast cancer xenograft mouse model demonstrated that TAN administration at 2.5 mg/kg significantly decreased tumor volume and tumor weight. Importantly, no noticeable differences in body weight were observed between the groups. These findings suggest that TAN may not induce systemic toxicity during the treatment period (Ko et al., 2020), supporting its anti-tumor activity in vivo. Collectively, these data support TAN as a therapeutic candidate that can suppress tumor growth without causing severe adverse effects, highlighting the need for further preclinical and clinical evaluation.

Colorectal Cancer

Previous studies have demonstrated that TAN exhibits synergistic anti-cancer effects with conventional chemotherapeutic agents in vitro, and this cooperative activity is also evident in vivo. In a xenograft model, the combined administration of TAN and 5-FU produced markedly greater suppression of tumor growth than the control group or either monotherapy alone. Furthermore, TAN treatment resulted in pronounced upregulation of phosphatase and tensin homolog (PTEN), accompanied by a significant reduction in phosphorylated AKT expression within tumour tissues. These results suggest that TAN may enhance the anti-tumour efficacy of 5-FU by augmenting PTEN-mediated negative regulation of the AKT oncogenic signalling pathway. Collectively, the anti-tumor effects of TAN observed in animal models support its potential for future clinical evaluation.

BIOAVAILABILITY AND METABOLISM OF TAN

TAN exhibits pronounced lipophilicity due to its multiple methoxy substitutions, a physicochemical property that markedly reduces its aqueous solubility and consequently limits its gastrointestinal absorption. In a pharmacokinetic study in rats, oral administration of TAN (50 mg/kg) yielded a T_max of 340.0 ± 48.99 min and a C_max of 0.87 ± 0.33 μg/mL, suggesting relatively slow absorption and limited systemic exposure following conventional oral dosing (Hung et al., 2018). These results indicate that a drug delivery system is needed to bring TAN into clinical application.

TAN exhibits metabolic biotransformation in vivo, which substantially influences its systemic exposure and biological fate. The primary metabolic pathways of TAN include demethylation, demethoxylation, hydroxylation, and remethoxylation, followed by phase II conjugation reactions such as glucuronidation and sulfation. These metabolic reactions generate multiple structurally modified metabolites with altered physicochemical properties. Cytochrome P450 enzymes, particularly CYP1A1 and CYP1B1, play a central role in TAN metabolism in the liver and extrahepatic tissues, catalyzing hydroxylation and demethylation reactions that yield metabolites such as 5-demethyltangeretin (5-DT) and 4′-hydroxy tangeretin (4′-OH TAN). In addition to hepatic metabolism, gut microbiota contribute to TAN biotransformation by producing secondary metabolites and by deconjugating glucuronide and sulfate conjugates, thereby affecting intestinal reabsorption and systemic availability (Guo et al., 2021; Surichan et al., 2018).

One of these metabolites, 5-demethyltangeretin (5-DT), exhibits anticancer activity in several cancer cells, inhibiting proliferation and inducing cell cycle arrest and apoptosis (Charoensinphon et al., 2013; Ma et al., 2014; Wu et al., 2020). These results suggest that the anticancer effects of TAN may be attributable not only by TAN but also by active metabolites generated through metabolic conversion. Therefore, further studies are needed to elucidate the mechanism of action, pharmacokinetics, and cancer type-specific anticancer activity of metabolites such as 5-DT derived from TAN metabolism.

LIMITATIONS AND FUTURE STUDIES

TAN exhibits low bioavailability and slow in vivo absorption. To address these limitations, previous research has explored advanced nano-formulation strategies to enhance its pharmacokinetic performance. Nano-enabled delivery systems such as polymeric nanoparticles, lipid nanoparticles, nanoliposomes, micellar systems, and self-nanoemulsifying drug delivery systems (SNEDDS) have been shown to substantially improve solubilization, confer physicochemical stability in gastrointestinal environments, augment interactions with intestinal epithelial membranes, facilitate lymphatic transport, and mitigate metabolic degradation (Bhardwaj et al., 2024; Mitchell et al., 2021; Porter et al., 2007). Previous studies have provided accumulating evidence that lipid-based nanocarriers enhance the solubilisation of TAN by mixed micelles and protect the compound from premature degradation, thereby improving its intestinal bioaccessibility (Chen et al., 2015; Huang et al., 2025). These studies suggest that delivery systems such as lipid-based nanosystems are essential for the oral formulation of TAN and provide a methodology to overcome the limitations of TAN. In addition to formulation-based approaches, structure-activity relationship (SAR)-guided structural optimization may also serve as a complementary strategy to overcome these intrinsic limitations. Strategic structural modifications, such as the introduction of polar substituents or heterocyclic moieties (e.g., thiazole or amine groups) into the pharmacophore, could improve aqueous solubility, metabolic stability, and target binding, thereby enhancing the anti-cancer efficacy of TAN (Fegade et al., 2025; Nath et al., 2025).

Because the anti-cancer effects of TAN have recently been discovered, the current body of preclinical and clinical evidence remains considerably limited. Phytochemicals are often considered to have relatively low toxicity, but toxicity studies on TAN are limited. Due to their highly fat-soluble nature, TAN can increase the hepatic burden and accumulate in fat-rich tissues. These potential risk factors emphasise the need for a systematic evaluation of TAN’s in vivo safety, long-term toxicity, and metabolic consequences. Previous studies have demonstrated that TAN can trigger a variety of biological responses, including anti-inflammatory, antioxidant, and anti-cancer activities, but these results alone are inadequate to advance TAN as a therapeutic candidate. Important pharmacologic parameters, including optimal dose range, frequency of administration, therapeutic range, and potential for drug interactions, remain to be elucidated. Therefore, more research is needed for reproducibility and clinical application.

In addition to these limitations, further mechanistic studies are required to elucidate the anti-cancer potential of TAN. Current research has mainly focused on apoptosis, autophagy, and cell cycle arrest. However, ferroptosis, an iron-dependent form of regulated cell death characterised by lipid peroxidation, has not been investigated. Because TAN modulates oxidative stress, it may also influence ferroptosis-related pathways, including the regulation of GPX4, the accumulation of lipid ROS, iron metabolism, and the system Xc⁻ signalling pathway (Li et al., 2022). Future studies for the role of ferroptosis in TAN-mediated anti-cancer effects could provide further mechanistic insights and expand TAN’s therapeutic potential. Collectively, additional studies on pharmacokinetics, safety, formulation optimisation, and the mechanisms underlying the anti-cancer effects of TAN are required to enable its clinical application as an anti-cancer therapy.

CONCLUSION

Flavonoids are a class of plant compounds with antioxidant, anti-inflammatory, anti-cancer, anti-bacterial, and anti-viral properties. Based on structural differences, flavonoids are classified into different subtypes: flavones, flavonols, and flavanones. TAN is a flavonoid that is mainly abundant in citrus peel and has a methylated flavone structure, which increases its biological activity and pharmacological stability. Previous studies have demonstrated that TAN induces cell-cycle arrest through the inhibition of CDK2 and various cyclin family proteins, while concurrently activating both extrinsic and intrinsic apoptosis pathways by increasing the expression of Fas and caspase-9. TAN also enhances the release of cytochrome c from mitochondria, thereby strengthening the intrinsic cell death pathway. In addition, TAN has been reported to potentiate the efficacy of chemotherapeutic agents and radiotherapy. These mechanistic features collectively indicate that TAN exerts broad inhibitory effects on cancer cell growth and survival, underscoring its multi-targeted anti-cancer properties. In addition to inducing apoptosis and cell cycle arrest, TAN suppresses EMT by regulating the expression of E-cadherin and N-cadherin, thereby reducing the migration and metastatic ability of cancer cells. TAN also impairs the differentiation and expansion of CSCs. Furthermore, in vivo studies have demonstrated that TAN reduces tumour weight and volume, as well as downregulating the expression of MMP-9 and PCNA. These findings support the potential of TAN as an adjuvant or monotherapy in cancer treatment.

However, several limitations remain regarding the therapeutic application of TAN, such as TAN’s high lipophilicity, poor bioavailability, and a short residence time in vivo. Therefore, the clinical application of TAN will require the development and implementation of advanced drug-delivery platforms, including polymeric nanoparticles, lipid nanoparticles, nanoliposomes, and micellar systems. Furthermore, comprehensive and systematic studies are required to fully characterize the metabolic pathways, tissue distribution, and potential in vivo toxicities of TAN. Nevertheless, TAN exhibits enhanced anti-cancer effects compared with other PMFs, such as NOB and SIN. Additionally, the distinctive structural features of TAN provide a significant advantage in repositioning as an anti-cancer agent. This review highlights the clinical potential of TAN and outlines the further research required to establish its credibility and effectiveness as an anti-cancer agent.