Nobiletin has been extensively studied for its ability to modulate several signaling pathways that are often dysregulated in cancer cells. For instance, nobiletin has been shown to modulate the Toll-like receptor (TLR) signaling pathway, which is implicated in prostate cancer progression, by potentially inducing the TLR3/interferon regulatory factor (IRF)3 signaling pathway and enhancing its activation when combined with polyinosinic:polycytidylic acid (Poly I:C). This suggests that nobiletin may mediate the TLR3 signaling pathway, with the combined treatment of nobiletin and Poly I:C showing a more marked effect in LNCaP cells than in prostate cancer-3 cells. This cell type-dependent response is noteworthy, as it indicates that the immunomodulatory effects of nobiletin may vary according to the genetic background or androgen receptor status of prostate cancer cells, offering potential for patient stratification in future therapeutic applications. Furthermore, the study indicates that while nobiletin can improve the activation of the TLR3/IRF3 signaling pathway (which promotes apoptosis), it also leads to a reduction in the activation of the TIR-domain-containing adapter-inducing interferon-β/receptor-interacting protein kinase 1/Fas-associated protein with death domain signaling pathway, highlighting the complex regulatory role of nobiletin in influencing apoptotic cell death in vitro (16).

Moreover, through affinity chromatography, proteomics, computer modeling and various biochemical analyses, Gao et al (29) identified heat shock cognate 70 (HSC70) as a novel direct protein target of nobiletin in colon cancer cells. In these laboratory experiments, nobiletin was found to bind to HSC70 at its ATP-binding site, thereby inhibiting its ATPase activity and disrupting cancer cell proliferation. Furthermore, the study revealed that the major colonic metabolites of nobiletin, which are generated in the colon of nobiletin-fed mice, produced similar inhibitory effects on HSC70-mediated pro-carcinogenic events as nobiletin itself.

In vivo studies have further confirmed the chemopreventative potential of nobiletin, showing reduced tumor incidence and progression in animal models of cancer (30). For example, nobiletin effectively inhibited the formation of metastatic lung nodules in nude mice (31). Chu et al (32) found that sweet orange peel extract and its bioactive compound nobiletin exhibit notable anti-proliferative effects against human hepatoma cells both in vitro and in vivo, potentially through inducing cell cycle arrest, apoptosis and the generation of ROS, which contribute to its antitumor activity. Additionally, Luo et al (33) observed a marked inhibitory effect of nobiletin on tumor growth in a nude mouse model of lung cancer (A549 ×enograft). These findings suggest that nobiletin induces p53-mediated cell cycle arrest and apoptosis by modulating the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 protein ratio. Ma et al (34) performed a series of in vitro and in vivo experiments and discovered that nobiletin caused G₂ cell cycle arrest and altered protein expression levels by reducing Bcl-2 and cyclooxygenase-2 (COX-2) while increasing Bax and caspase-3 levels in SMMC-7721 cells. In vivo, nobiletin was shown to inhibit H22 tumor growth, significantly downregulating the expression of COX-2, Bax and caspase-3, and lower Bcl-2/Bax ratios, demonstrating its substantial inhibitory impact on hepatocellular carcinoma in both settings.

Nobiletin regulates protein expression which in turn affects the cell cycle, and it also has direct effects on cell cycle progression. The two processes are interconnected rather than separate. For example, Zhang et al (46) demonstrated that nobiletin markedly inhibits the proliferation of human pancreatic cancer cells (PANC-1) in vitro by inducing apoptosis and cell cycle arrest. The study revealed that nobiletin upregulated the expression of the proapoptotic protein Bax and downregulated the antiapoptotic proteins Bcl-2 and p53, leading to an accumulation of cells in the G₀/G₁ phase and a decrease in the S phase. Moreover, Jiang et al (47) reported that nobiletin inhibits the growth of multidrug-resistant SKOV3/TAX ovarian cancer cells by inducing G₀/G₁ cell cycle arrest and reducing G₂/M phase transition, accompanied by upregulation of p53 and p21 proteins (47). Nobiletin also promoted apoptosis in multidrug-resistant SKOV3/TAX ovarian cancer cells via the intrinsic apoptotic pathway, as evidenced by increased levels of cleaved caspase-9/-3 and poly(ADP-ribose) polymerase (PARP). Furthermore, nobiletin disrupted autophagic degradation in these cells, leading to autophagic flux blockade, which in turn amplified its apoptotic effect. A key mechanistic finding from this study was that nobiletin stimulated the AKT signaling pathway, and this activation was implicated in both the suppression of autophagic degradation and the enhancement of apoptosis (47).

Nobiletin exerts its antitumor effects through a complex interplay with multiple signaling pathways that regulate the cell cycle (48). For example, Cheng et al (41) revealed that nobiletin exerted its anti-metastatic effects on human osteosarcoma U2OS and HOS cells by downregulating the expression of matrix metalloproteinases (MMP)-2 and MMP-9. This was achieved through the inhibition of the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, leading to the inactivation of downstream transcription factors, including NF-κB, cAMP response element-binding protein (CREB) and specificity protein 1 (SP-1), thereby suppressing cellular motility, migration and invasion. Lien et al (49) found that nobiletin treatment led to a reduction in cell viability and cell cycle arrest at the G₀/G₁ phase in human U87 and Hs683 glioma cell lines. Western blotting data revealed that nobiletin attenuated the expression of cyclin D1, cyclin-dependent kinase (CDK)2, CDK4 and E2F1, as well as the phosphorylation of AKT and MAPKs, including p38, ERK and JNK. Furthermore, nobiletin was shown to inhibit glioma cell migration, as demonstrated by functional wound healing and Transwell migration assays. Wei et al (50) demonstrated that nobiletin exerts its antitumor effects on renal carcinoma cells through the inhibition of the proto-oncogene tyrosine-protein kinase Src (SRC)/AKT/signal transducer and activator of transcription (STAT)3/YY1-associated protein 1 (YY1AP1) signaling pathway, leading to cell cycle arrest in the G₀/G₁ phase and the promotion of apoptosis. The study showed that nobiletin decreased the nuclear localization of STAT3 and YY1AP1, and reduced the levels of phosphorylated (p-)SRC, AKT and STAT3, while increasing the phosphorylation of YY1AP1, suggesting a potential novel therapeutic strategy for kidney cancer treatment by targeting these signaling components.

Nobiletin also modulates protein expression to exert its anticancer effects. For example, Chen et al (51) demonstrated that nobiletin inhibits the growth and proliferation of human acute myeloid leukemia (AML) cells by downregulating the expression of the KIT proto-oncogene, receptor tyrosine kinase (c-KIT) gene, which is crucial for leukemia progression. Nobiletin treatment resulted in a marked reduction of c-KIT mRNA and protein levels in AML cells, confirmed by the inhibition of KIT promoter activity. The anti-leukemic effects of nobiletin were abolished by the overexpression of c-KIT, indicating that nobiletin exerts its anticancer effects through the downregulation of c-KIT gene expression. In a previous study, it was found that nobiletin and its colonic metabolites, nobiletin-Met, notably suppressed colitis-associated colon carcinogenesis by downregulating inducible nitric oxide synthase (iNOS) and upregulating the nuclear factor erythroid 2-related factor (Nrf2) signaling pathway, which is associated with the induction of antioxidative enzymes and cell cycle arrest (52). Jiang et al (53) demonstrated that nobiletin suppressed the growth and metastasis of MIAPaCa-2 pancreatic cancer cells by triggering autophagy (increased LC3-II/I and decreased p62), arresting the cell cycle at G₀/G₁ (downregulating cyclin D1 and CDK4) and blocking NF-κB signaling in a concentration-dependent manner, thereby inhibiting proliferation, migration and metastasis. Turdo et al (54) demonstrated that nobiletin selectively targets colorectal cancer stem cells (CSCs) and synergizes with 5-fluorouracil plus oxaliplatin (FOX). Nobiletin reduces stem cell viability by arresting the cell cycle at S and G₂/M phases and upregulating pro-apoptotic genes; it disrupts CSC maintenance by downregulating Wnt signaling, cluster of differentiation (CD)44v6 expression and clonogenic capacity, thereby reversing chemoresistance and inhibiting metastatic expansion.

Moreover, nobiletin has been found to influence the expression of miRNAs. miRNAs serve a crucial role in lung cancer by regulating key cellular processes and signaling pathways involved in tumorigenesis, including cell proliferation, differentiation, angiogenesis, apoptosis, invasion and metastasis, making them potential therapeutic targets and biomarkers in non-small cell lung cancer (NSCLC) (55). For example, Han et al (42) demonstrated that nobiletin repressed miR-15-5p, thereby relieving inhibition of the negative WNT regulators naked cuticle 1, axin 2 and Wnt inhibitory factor 1. This cascade reduced nuclear β-catenin and its transcriptional targets (c-Myc, c-Jun and cyclin D1), abrogating stem-like properties, colony formation and inducing apoptosis. These findings establish nobiletin as a miRNA-mediated WNT pathway modulator in NSCLC. Jahan et al (56) explored the neuroprotective potential of nobiletin in human neural progenitor cells, revealing that nobiletin treatment notably restores deregulated miRNAs and proteins induced by sodium arsenate exposure, which are associated with neurodegeneration, oxidative stress response and apoptotic processes. This restoration suggests that the neuroprotective effects of nobiletin may be mediated through the modulation of key signaling pathways, including p53 and Wnt, which are implicated in cell death and cell cycle regulation.

Multiple independent studies across various cancer models consistently confirm that nobiletin targets key oncogenic pathways, including PI3K/AKT and MAPK, to block cancer cell expansion. However, emerging mechanisms such as epigenetic regulation and cancer stem cell targeting are supported by a more limited number of studies and require further validation (57). The broad-spectrum growth-inhibitory effects of nobiletin highlight its promise as a multi-mechanistic anticancer agent (58). For example, in a pulmonary carcinogenesis mouse model, oral administration of nobiletin markedly suppressed lung tumorigenesis, as evidenced by reduced tumor volume and attenuated cell proliferation. Furthermore, the effects were associated with notable cell cycle arrest, cellular apoptosis and the modulation of key proteins involved in cell proliferation and death, such as p21, cyclin B1, CDK1, cyclin D1, CDK6, CDK4, Bax, cleaved caspase-1 and cleaved PARP (59). Wang et al (14) elucidated the role of nobiletin in breast cancer cells, demonstrating its ability to inhibit cell proliferation in a dose-dependent manner and induce both apoptosis and pyroptosis. The research identified miR-200b as a key mediator of these effects, showing that nobiletin enhances miR-200b-induced pyroptosis and that JAZF zinc finger 1 (JAZF1) is a direct target of miR-200b. Furthermore, it revealed that nobiletin exerts its antitumor effects through the miR-200b/JAZF1/NF-κB signaling axis. The study by Gutiérrez-Venegas and Rosas-Martínez (60) showed that nobiletin and its derivative 5-demethylnobiletin can ameliorate hypopharyngeal squamous cell carcinoma by suppressing transforming growth factor β (TGF-β)-mediated EMT. The study indicated that these flavonoids inhibit EMT by reversing TGF-β-induced morphological changes, migration and the expression of EMT markers, suggesting their potential as therapeutic agents for the treatment of oral squamous cell carcinoma.

Autophagy is a double-edged sword in the context of cancer. While it can be a protective mechanism under stress conditions, it can also be hijacked by cancer cells to promote their survival and progression (61). The balance of autophagy is therefore a critical factor in cancer development and treatment response (62). Inhibiting autophagy has been shown to induce cell death in certain cancer types, including pancreatic (46) and ovarian (47) cancer, and to improve the outcomes of standard cancer therapies (63). Conversely, promoting autophagy in cancer cells could be a potential therapeutic strategy to weaken tumor resistance and enhance treatment efficacy (64). Understanding the complex interplay between autophagy and cancer is essential for developing novel cancer treatments that target this cellular process effectively. Moon and Cho (65) demonstrated that nobiletin activates a complex cellular response in SNU-16 human gastric cancer cells, involving the induction of glucose-regulated protein 78 kDa (GRP78) and other endoplasmic reticulum stress-related proteins, such as inositol requiring enzyme 1-α, activating transcription factor 4 and C/EBP homology protein (CHOP), leading to endoplasmic reticulum stress-mediated apoptosis. Additionally, the study found that nobiletin triggers protective autophagy, which when inhibited by chloroquine markedly enhances apoptosis, suggesting that nobiletin-induced apoptosis in gastric cancer cells is mediated through endoplasmic reticulum stress and autophagy pathways. Moreover, PI3K acts as a molecular switch that converts tumor-associated macrophages from an immune-suppressive to an immune-stimulatory state, thereby unleashing CD8⁺ T-cell cytotoxicity and enhancing antitumor immunity (66).

Chen et al (27) recently elucidated a lipid-metabolism-dependent mechanism by which nobiletin kills gastric cancer cells. The study showed that nobiletin directly binds sterol regulatory element-binding protein 1 (SREBP1), prevents its nuclear translocation and blocks SREBP1 occupancy on the ATP citrate lyase (ACLY) promoter. This leads to downregulation of ACLY-dependent fatty-acid synthesis and simultaneously inactivates the PI3K/AKT/mechanistic target of rapamycin (mTOR) axis. The resulting metabolic stress converts cytoprotective autophagy into autophagy-dependent cell death, an effect validated in a patient-derived xenograft model where nobiletin reduced tumor volume while suppressing SREBP1 nuclear accumulation and PI3K/AKT/mTOR signaling.

Nobiletin has emerged as a potent inhibitor of cancer cell invasion and migration (67). For example, research has found that nobiletin can affect the migration and invasion of hypopharyngeal squamous cell carcinoma (68). Nobiletin has been demonstrated to inhibit cancer cell invasion and migration by suppressing the activation of kinases that promote cell proliferation and survival, thereby reducing the downstream signaling involved in these processes (69). For example, Wu et al (58) revealed that nobiletin restrains breast cancer metastasis by blocking IL-6-driven ERK-STAT and JNK cascades. In MCF-7 and T47D cells, nobiletin dose-dependently suppressed phosphorylation of ERK1/2 and JNK, interrupting the downstream STAT and c-JUN transcriptional activity that underpins cell migration and invasion. Molecular docking analysis confirmed high-affinity binding of nobiletin to PI3K (−8.0 kcal/mol). Consistent with these in vitro findings, nobiletin reduced liver metastasis in xenograft models, underscoring its potential as a multi-pathway inhibitor against breast cancer dissemination. Liu et al (70) demonstrated that nobiletin inhibits EMT in renal carcinoma cells by inactivating the NF-κB and Wnt/β-catenin signaling pathways, thereby preventing hypoxia-induced migration and invasion, suggesting its potential as an anticancer agent in renal cell carcinoma.

The anti-invasive and anti-migratory effects of nobiletin are mediated through the modulation of several key signaling pathways and the regulation of MMPs (71). MMPs serve a crucial role in cancer progression by degrading the extracellular matrix, promoting the invasion and metastasis of cancer cells (72). For example, nobiletin inhibits MMP-9 enzymatic activity in retinal Müller cells by suppressing MMP-9 gene transcription and enhancing TIMP metallopeptidase inhibitor 1 (TIMP-1) production, which is mediated by the PI3K/AKT signaling pathway (73). This signaling pathway involves the participation of NF-κB, STAT3, ERK (74), JNK/SAPK, p38 MAPK and AKT/Nrf2 (75), which are modulated by nobiletin to inhibit cancer cell invasion (76). For example, Xu et al (77) revealed that nobiletin suppresses renal carcinoma by simultaneously blocking the Janus tyrosine kinase 2 (JAK2)/STAT3 and PI3K/AKT cascades. Exposure to nobiletin dose-dependently reduced phosphorylation of JAK2, STAT3, PI3K and AKT, thereby silencing downstream pro-survival signals and upregulating apoptotic proteins. This dual-pathway inhibition arrested cell cycle progression, curtailed migration and invasion, and triggered apoptosis in 786-O and ACHN cell lines. The PI3K/AKT pathway, which primarily affects distant metastasis by reducing tumor cell adhesion, is also modulated by nobiletin; it downregulates the expression of MMP-1 and MMP-9, and upregulates TIMP-1, thus inhibiting the invasion of cancer cells. In liver cancer, nobiletin reduces the levels of p-ERK2 and p-AKT, inhibiting cell metastasis involving ERK and PI3K/AKT pathways induced by hepatocyte growth factor (78) (Table I) (14,16,21,27,29,33,34,41,46,47,49–53,58,65,70,74).

Nobiletin also inhibits tumor angiogenesis in breast cancer cells by targeting the p38 mitogen-activated protein kinase, NF-κB and Nrf2 pathways. A previous study showed that nobiletin treatment led to decreased cell viability and induced apoptosis in MCF-7 cells, while also inhibiting cell migration by downregulating MMP-2 and MMP-9 expression (79). Additionally, in the TGF-β1/Smad3 signaling pathway, nobiletin inhibits EMT, a cellular process essential during cancer metastasis. It does so by inhibiting the expression of MMP-2, MMP-9, p-SRC, p-focal adhesion kinase (FAK), p-paxillin, Snail, Slug, Twist and zinc finger E-box-binding homeobox 1, thereby inhibiting the EMT of human NSCLC cells (31). The ERK/JNK pathway is another target of nobiletin, which inhibits the activities of ERK and JNK, decreasing the mRNA expression and protein levels of MMP-2 and MMP-9, and inhibiting the DNA binding activity of transcription factors NF-κB, CREB and SP-1 in tumor cells (41). Moreover, in the AKT/glycogen synthase kinase-3β (GSK-3β)/β-catenin signal pathway, nobiletin has been shown to enhance chemosensitivity to Adriamycin in A549 NSCLC cells by modulating key components of this pathway (Fig. 2). Moon et al (80) demonstrated that nobiletin treatment decreased the expression of multidrug resistance-associated protein 1 (MRP1) and neuroblastoma-derived MYCN proto-oncogene bHLH transcription factor (MYCN), as well as downregulating AKT, GSK-3β and β-catenin. This regulation led to the accumulation of intracellular Adriamycin and enhanced apoptosis in A549/ADR cells.

Beyond its anti-metastatic effects, nobiletin has demonstrated the ability to overcome multiple modes of drug resistance through distinct mechanisms. In efflux-mediated resistance, nobiletin downregulates MRP1 expression via the AKT/GSK-3β/β-catenin/MYCN pathway, increasing intracellular accumulation of chemotherapeutic agents, such as Adriamycin, in lung cancer cells (80). Regarding EMT-associated resistance, nobiletin reverses hypoxia-induced EMT by inactivating Notch-1 signaling and activating miR-200b, thereby restoring sensitivity to EGFR inhibitors in lung cancer models (45). For CSC-related resistance, nobiletin selectively targets colorectal CSCs by downregulating Wnt signaling and CD44v6 expression, reversing chemoresistance to FOX (54). Additionally, emerging evidence suggests nobiletin may counteract immune escape by modulating PI3Kγ signaling, which converts tumor-associated macrophages from an immunosuppressive to an immunostimulatory phenotype, thereby enhancing CD8⁺ T-cell cytotoxicity (66).

Tumor angiogenesis, a critical process in the advancement of cancer, involves the development of new blood vessels to support the growing tumor (81). This process is initiated as a response to inadequate blood circulation, ensuring that the tumor receives the necessary oxygen and nutrients required for its expansion (82). Additionally, tumors release multiple angiogenic factors that stimulate the formation of these new blood vessels (83). Among these factors, vascular endothelial growth factor (VEGF) stands out as a notable contributor to the angiogenic activity associated with tumor growth (84). Kisacam (85) has shown that nobiletin exerts its anticancer effects in colon cancer cells through multiple pathways. Specifically, it inhibits AKT activity, which is crucial for cancer cell survival and angiogenesis, by targeting Bax, Bcl-2 and p70S6K at concentrations higher than 100, 500 and 1,000 µM, respectively. Nobiletin also modulates the levels of angiogenic factors VEGF and MMP-7, as well as cell cycle regulators 4EB1 and tubulin. Its ability to suppress AKT signaling and subsequently reduce angiogenic factors and regulators suggests that nobiletin could be an effective preventive agent against colon cancer progression by targeting these key pathways.

Additionally, STAT3 is involved in the regulation of various cellular processes, including cell survival, proliferation and differentiation (86). FAK is another key player in pancreatic cancer progression, as it contributes to cell adhesion, migration and invasion, which are critical steps in cancer metastasis. Inhibition of FAK can lead to compensatory activation of STAT3 signaling, which may promote cancer cell survival and resistance to treatment (87). Sp et al (88) identified a novel mechanism by which nobiletin inhibits tumor angiogenesis in estrogen receptor-positive (ER⁺) breast cancer cells. The study revealed that nobiletin suppresses angiogenesis by targeting the Src/FAK/STAT3 signaling pathway through paxillin (PXN), leading to the inhibition of angiogenic markers and reduced cell migration and invasion. In another study by Sp et al (88), nobiletin was found to inhibit angiogenesis in ER⁺ breast cancer cells by regulating the Src/FAK/STAT3 signaling pathway through PXN, which is a critical mediator of angiogenic activity. The research demonstrated that nobiletin treatment led to a marked reduction in angiogenesis markers, as evidenced by western blotting and quantitative PCR analysis, and further confirmed its anti-angiogenic activity in human umbilical vein endothelial cells. Additionally, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay results indicated that nobiletin inhibits STAT3/DNA binding activity and STAT3 binding to a novel binding site of the PXN gene promoter. Moreover, CD36, a protein with notable implications in tumor angiogenesis, is involved in the metabolism of fatty acids and thus influences the metastatic spread of cancer. A study demonstrated that nobiletin inhibits CD36-dependent tumor angiogenesis, migration, invasion and sphere formation through the CD36/STAT3/NF-κB signaling axis, providing a detailed mechanism of action (89). The study identified a g-interferon activation site element in the CD36 gene promoter that acts as a STAT3 binding site, confirmed by a ChIP assay, and showed that nobiletin also acts through the CD36/STAT3/NF-κB signaling axis. These findings suggest that nobiletin exerts its inhibitory effects on tumor angiogenesis by modulating CD36 signaling, which is crucial for tumor progression (90).

Yang et al (24) demonstrated that nobiletin exerted notable effects on the MAPK/NF-κB signaling pathway in the synovial membrane of rats with arthritis induced by collagen. The study showed that nobiletin treatment led to a marked reduction in angiogenesis and inflammatory infiltration within the synovial tissue, as well as decreased levels of key pro-inflammatory cytokines, such as IL-1β, monocyte chemotactic protein-1, IL-6 and tumor necrosis factor-α, in rats with collagen-induced arthritis. Additionally, the protein expression levels of p-p38, p-p65 and NF-κBα were downregulated by nobiletin, indicating its inhibitory role in the p38/NF-κB signaling pathway. Deng et al (91) reported a novel mechanism by which nobiletin exerts its anti-atherosclerotic effects, involving activation of PINK1/Parkin-mediated mitophagy and suppression of the NLRP3 inflammasome signaling pathway. The study demonstrated that nobiletin modulates blood lipid metabolism and inflammation in mice, inhibiting arterial plaque formation. Notably, nobiletin was found to activate Parkin-ubiquitin kinase 1/Parkin-mediated mitophagy and suppress nucleotide-binding oligomerization domain 3 inflammasome production.

Apoptosis, a programmed cell death process, serves a pivotal role in maintaining tissue homeostasis and is frequently disrupted in cancer development. The induction of apoptosis in tumor cells by chemopreventative agents represents a promising strategy for cancer treatment (92). Previous studies have consistently shown that nobiletin treatment leads to a notable increase in apoptotic cell death, which is characterized by caspase activation (93), DNA fragmentation and cell membrane blebbing (94). For example, Zhang et al (95) revealed that nobiletin treatment led to a decrease in cell viability and cell membrane integrity, increased expression of hypoxia inducible factor 1α, cell cycle arrest in the G₁ phase and notable cell apoptosis in human choriocarcinoma JEG-3 cells. The protective effects of nobiletin were associated with its ability to modulate the p53 signaling pathway, as evidenced by the downregulation of the mRNA and protein levels of p53 and p21, and an upregulation of the Bcl-2/Bax mRNA and protein ratio in JEG-3 cells.

Nobiletin has been shown to cause the release of cytochrome c from the mitochondria, leading to the activation of caspase-9 and subsequent cleavage of caspase-3, which are critical executioner caspases in the apoptotic process (96). This effect is often accompanied by a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bax (97). For example, Li et al (98) demonstrated that nobiletin overcomes oxaliplatin resistance in colorectal cancer by simultaneously promoting apoptosis and suppressing the PI3K/AKT/mTOR axis. Co-treatment with nobiletin upregulated pro-apoptotic Bax and cleaved-caspase-3 while downregulating anti-apoptotic Bcl-2 in colorectal cancer cells (HCT116 and SW480), thereby amplifying oxaliplatin-induced cell death. Concurrently, nobiletin attenuated PI3K/AKT/mTOR signaling, a pathway frequently hyper-activated in drug-resistant tumors, leading to reduced cell survival and enhanced chemosensitivity. Additionally, nobiletin has been reported to trigger cancer cell apoptosis through caspase-3 activation and PARP cleavage in lung cancer cells (A549), and may serve as a key regulator in mitigating drug resistance by modulating various signaling pathways (Fig. 3), positioning it as a promising therapeutic agent for lung cancer treatment (25).

The molecular mechanisms underlying nobiletin-induced apoptosis appear to be multifaceted and involve the modulation of several key signaling pathways. One of the primary pathways affected by nobiletin is the mitochondrial apoptotic pathway (99). Zhang et al (100) demonstrated that nobiletin markedly inhibits cell proliferation and induces DNA damage in ovarian cancer cells in a dose-dependent manner, leading to apoptosis through the elevation of PARP levels. Additionally, the study revealed that nobiletin decreases mitochondrial membrane potential, triggers ROS generation and induces autophagy, which collectively contribute to gasdermin D-mediated pyroptosis in human ovarian cancer cells. These findings suggest that nobiletin has a dual role in promoting apoptosis and triggering ROS-mediated pyroptosis through the regulation of autophagy. Luo et al (33) showed that nobiletin dose-dependently suppresses the proliferation of A549 cells and induces apoptosis, as evidenced by DNA fragment and comet assays. Additionally, flow cytometric analysis revealed that nobiletin causes cell cycle arrest at the G₂/M phase. Western blot analysis indicated that nobiletin pretreatment led to decreased Bcl-2 and increased Bax protein expression in A549 cells, which was associated with elevated p53 expression compared with that in the controls. The study also observed an overt inhibitory effect of nobiletin on tumor growth in a nude mouse xenograft model of lung cancer (A549 cells) in vivo. Chen et al (101) demonstrated that nobiletin suppresses gastric cancer by directly inhibiting ACLY, a key enzyme in fatty acid synthesis. This inhibition reduces neutral lipid accumulation and activates the IRE-1α/GRP78/CHOP-mediated endoplasmic reticulum stress pathway, leading to apoptosis. ACLY overexpression abolished these effects, confirming strict ACLY dependence. In vivo, nobiletin similarly suppressed tumor growth in a patient-derived xenograft (PDX) model of gastric cancer by disrupting de novo fatty acid synthesis, positioning it as a novel ACLY inhibitor for gastric cancer therapy.

While the preceding sections have delineated nobiletin's modulation of individual signaling cascades, its true therapeutic potential as a multi-target agent lies in its ability to simultaneously influence the complex web of interconnected pathways that govern cancer cell behavior. One of the most clinically relevant interactions involves the PI3K/AKT and NF-κB pathways. AKT, a downstream effector of PI3K, can directly activate IκB kinase, leading to IκBα phosphorylation and degradation, thereby releasing NF-κB for nuclear translocation and transcriptional activation of pro-survival genes (98). Nobiletin's concurrent suppression of both PI3K/AKT phosphorylation and NF-κB nuclear translocation, observed in colorectal cancer and pancreatic cancer models, suggests that it may disrupt this positive feedback loop, thereby amplifying its apoptotic effects (53). The Wnt/β-catenin pathway, critical for stemness and proliferation, exhibits bidirectional crosstalk with the MAPK/ERK cascade. ERK can phosphorylate LDL receptor-related protein 6, a Wnt co-receptor, enhancing Wnt signaling, while β-catenin can transcriptionally regulate components of the MAPK pathway (102). In NSCLC, nobiletin was shown to simultaneously downregulate nuclear β-catenin (via miR-15-5p-mediated derepression of Wnt inhibitors) and suppress ERK phosphorylation, potentially disrupting this synergistic interaction to abrogate stem-like properties and colony formation (42).

Emerging evidence suggests that nobiletin may target master upstream regulators that orchestrate multiple downstream pathways. For instance, the inhibition of PI3K by nobiletin, confirmed by molecular docking showing high-affinity binding to the PI3K active site, can simultaneously attenuate both AKT/mTOR and downstream NF-κB signaling, positioning PI3K as a hierarchical node in the multi-target action of nobiletin (58). Similarly, the direct binding of nobiletin to HSC70, a chaperone protein involved in stabilizing multiple oncogenic kinases, may represent an even more proximal mechanism that explains its broad-spectrum pathway inhibition (29).

The relative contribution of each pathway to the effects of nobiletin varies considerably by tumor type and genetic background. In KRAS-mutant pancreatic cancer, nobiletin appears to primarily exert its effects through MAPK pathway suppression and autophagy modulation, with NF-κB inhibition playing a secondary role (54). By contrast, in triple-negative breast cancer, the miR-200b/JAZF1/NF-κB axis emerges as the dominant mechanism, with PI3K/AKT modulation contributing to a lesser extent (14). In colorectal cancer, the HSC70 interaction and subsequent destabilization of multiple client proteins may represent the primary initiating event, with downstream pathway inhibition (including PI3K/AKT and Wnt/β-catenin) occurring as secondary consequences (54). These context-dependent differences likely reflect variations in pathway addiction, mutational landscape and tumor microenvironment composition across cancer types.