Molecular Mechanisms of the Anticancer Activity of the Flavonoid Kaempferol: A Comprehensive Review

Abstract

Kaempferol (KAE), a natural flavonoid, has emerged as a promising multi-target antineoplastic agent characterized by high efficacy and minimal systemic toxicity. Moving beyond fragmented descriptive summaries, this comprehensive review provides a highly integrated conceptual framework of KAE’s anticancer mechanisms. Specifically, KAE orchestrates tumor eradication by enforcing cell cycle arrest across multiple phases and triggering a complex, interconnected network of programmed cell death. We highlight how reactive oxygen species and endoplasmic reticulum stress serve as central upstream nodes driving the mechanistic crosstalk among apoptosis, lethal autophagy, gasdermin E-mediated pyroptosis, and ferroptosis. Furthermore, KAE actively remodels the tumor microenvironment by inhibiting angiogenesis and repolarizing tumor-associated macrophages, thereby converting immunosuppressive "cold" tumors into immune-active "hot" tumors. Notably, this review introduces the emerging prebiotic-like crosstalk between KAE and the gut microbiome, providing a strong mechanistic rationale for its synergistic application with immune checkpoint inhibitors. As a potent chemosensitizer, KAE also overcomes multidrug resistance and mitigates chemotherapy-induced toxicities. Finally, we critically evaluate current translational bottlenecks—including the disparity between supraphysiological in vitro concentrations and clinical pharmacokinetics, the lack of robust in vivo validations, and the long-term biosafety of emerging nano-delivery systems. By addressing these critical limitations, this review offers strategic perspectives to bridge the gap from preliminary bench research to future precision oncological practice.

INTRODUCTION

Cancer remains the second leading cause of death worldwide [1]. Its initiation and progression are driven by complex pathological events, notably unconstrained cell proliferation, evasion of programmed cell death, and dynamic alterations within the tumor microenvironment (TME) [2]. Given that conventional treatments, such as chemotherapy and targeted therapy, are frequently associated with severe drug resistance and systemic toxicity, there is an urgent clinical need for safe and effective intervention strategies.

Traditional Chinese medicine (TCM) in the treatment of malignant tumors is rooted in the holistic concept of "strengthening body resistance and eliminating pathogens" (Fuzheng Quxie). TCM has demonstrated unique advantages in reducing toxicity and enhancing therapeutic efficacy by synergistically modulating the tumor immue microenvironment (TIME) through multi-target interventions [3]. Within this context, kaempferol (KAE), a representative flavonol compound, has emerged as a focal point in integrative oncology due to its ability to precisely modulate molecular signaling pathways and induce tumor cell death with high efficacy and low toxicity.

Chemically identified as 3,4',5,7-tetrahydroxyflavone, KAE is widely distributed in various TCMs, such as Radix Astragali (Huangqi), Ginkgo biloba L., and Forsythia suspensa, as well as in homologous medicinal and edible plants like leeks and strawberries. Modern pharmacological research has confirmed that KAE not only possesses significant biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects [4], but also exhibits broad-spectrum anti-tumor potential in endometrial, hepatic, lung, nasopharyngeal, and gastrointestinal malignancies [5-9]. Its anti-tumor efficacy does not rely on a single pathway; instead, it is exerted through multidimensional synergies, including the induction of cell cycle arrest, programmed cell death, and the remodeling of the TME. Focusing on these three pivotal areas, this article systematically summarizes the research progress on the molecular mechanisms of KAE’s anti-tumor effects over the past decade (Fig. 1), aiming to provide a comprehensive scientific reference for its clinical translation and drug development.

Figure 1. A comprehensive conceptual framework of the multifaceted anticancer activities and translational potential of KAE.

The central chemical structure represents the natural flavonoid KAE. The lower panel delineates the four core anti-tumor mechanistic pillars targeted by KAE: (1) induction of cell cycle arrest across various phases, (2) triggering of extensive programmed cell death networks, (3) structural and immune remodeling of the TME, and (4) the emerging bidirectional crosstalk with the gut microbiome. The upper panel highlights the translational application of KAE in combination therapy paradigms, demonstrating its capacity to synergize with classical chemotherapeutic agents (e.g., Docetaxel, Doxorubicin, 5-FU) and multidrug resistance modulators (e.g., Verapamil) to enhance clinical efficacy and overcome chemoresistance. KAE, kaempferol; 5-FU, 5-fluorouracil; TME, tumor microenvironment; G1, gap 1 phase; S, synthesis phase; G2, gap 2 phase; M, mitosis phase; GSDME, gasdermin E.

It is important to distinguish natural KAE (3,4',5,7-tetrahydroxyflavone) from its structurally related flavonol analogs (e.g., quercetin or luteolin) and synthetically modified derivatives. While natural KAE exhibits broad-spectrum anti-tumor activities as primarily discussed herein, certain cited studies—particularly recent in silico molecular docking models—also explore novel KAE derivatives engineered for enhanced specific target affinity (e.g., against CDK2). To avoid potential ambiguity, throughout this review, the abbreviation "KAE" refers strictly to the natural parent compound unless explicitly designated as a "derivative".

While previous reviews have broadly summarized the general properties of flavonoids, this manuscript distinguishes itself by providing a highly integrated conceptual framework. We systematically delineate the complex crosstalk between canonical apoptosis and emerging, non-canonical cell death pathways, particularly ferroptosis and pyroptosis. More importantly, rather than merely offering descriptive summaries, this review critically evaluates the translational bottlenecks of KAE—specifically addressing the disparity between supraphysiological in vitro concentrations and clinical pharmacokinetic realities. By highlighting cutting-edge synergistic strategies, including nanomedicine applications, gut microbiome remodeling, and synergy with immune checkpoint inhibitors, this review aims to bridge the critical gap between preliminary laboratory findings and future oncological practice (Fig. 1).

REGULATION OF CELL CYCLE

The growth, metastasis, and drug sensitivity of tumors are intricately linked to complex cell cycle regulation [10]. The cell cycle consists of G1, S, G2, and M phases, which are precisely monitored by three core checkpoints: G1/S, G2/M, and the spindle assembly checkpoint [11]. The progression of this cycle is driven by the coordinated actions of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors. Under physiological conditions, cyclins bind to and activate specific CDKs, which then phosphorylate downstream target proteins to propel the cycle forward. However, in tumor cells, the dynamic homeostasis between these positive drivers and endogenous inhibitors (such as p21 and p27) is disrupted. The aberrant activation of CDKs leads to the characteristic uncontrolled and infinite proliferation of cancer cells [12].

Induction of G0/G1 phase arrest

Cell cycle checkpoints are essential for maintaining the orderly progression of the cell cycle and ensuring genomic stability during cell division. The primary function of the G1/S checkpoint is to monitor DNA integrity and determine whether the cell is prepared to enter the S phase. KAE exerts a "dual-action" inhibitory effect by downregulating the expression of Cyclin D1, thereby disrupting the CDK4/6/Cyclin D1 signaling axis. Concurrently, it upregulates the expression of the cyclin-dependent kinase inhibitors p21 and p27. This synergistic modulation prevents cells from bypassing the G1/S checkpoint, ultimately leading to cell cycle arrest in the G0/G1 phase [13].

Induction of S phase arrest

The S phase is dedicated to DNA replication, a process critically governed by the Cyclin A/CDK2 complex. Recent evidence indicates that KAE effectively halts cell cycle progression at this stage by directly targeting this regulatory machinery. Specifically, in vitro studies in colon cancer models confirm that KAE significantly downregulates the protein expression of both Cyclin A and CDK2 [14]. Furthermore, in silico computational modeling reveals that KAE exhibits exceptionally high binding affinity for the CDK2 active site, forming stable complexes that competitively inhibit its kinase function [15].

By concurrently suppressing the expression and physically obstructing the activity of the Cyclin A/CDK2 complex, KAE severely disrupts DNA synthesis, culminating in a distinct S phase arrest. This S-phase blockade has also been consistently observed in bladder cancer cells following KAE treatment [16]. Interestingly, KAE-induced arrest exhibits remarkable cell-type specificity; for instance, it predominantly induces G1 arrest in androgen-dependent 22Rv1 cells, but effectively triggers S and G2 phase arrest in androgen-independent PC-3 prostate cancer cells [17].

Induction of G2/M phase arrest

The G2/M checkpoint primarily monitors DNA damage repair and coordinates the preparation for mitosis. The pivotal transition into mitosis is strictly governed by the activation of the Cdc2 (CDK1)/Cyclin B1 complex, which inherently relies on the dephosphorylation action by the Cdc25C phosphatase. KAE has been robustly shown to interfere with this critical node. For instance, mechanistic studies reveal that KAE significantly decreases the protein levels of Cdc25C, Cdc2, and Cyclin B1, thereby robustly inhibiting the kinase activity of the Cdc2/Cyclin B1 complex and resulting in profound G2/M arrest [14].

Beyond gastrointestinal models, KAE induces G2/M cell cycle arrest across various tumor types through distinct signaling pathways. In SK-HEP-1 human hepatocellular carcinoma cells, KAE prevents the formation of the CDK1/Cyclin B complex to inhibit mitosis [18]. Furthermore, in HCCLM3 and Huh7 hepatoma cells, KAE triggers G2/M arrest and promotes apoptosis by modulating the ATM/CHEK2/KNL1 pathway [19]. In human ovarian cancer A2780/CP70 cells, G2/M arrest is mediated via the Chk2/Cdc25C/Cdc2 and Chk2/p21/Cdc2 signaling axes [20]. Similarly, KAE exhibits consistent G2/M-blocking efficacy in cholangiocarcinoma (RMCCA-1 and HuCCT-1) and renal cell carcinoma (786-O and 769-P) cell lines [21,22]. These effects are summarized in Table 1 [13,14,16-22].

Table 1.

Summary of cell cycle arrest induced by KAE in various human cancer cell lines

Cancer type	Cell line	Cell cycle arrest	References
Gallbladder cancer	GBC-SD, SGC996	G0/G1	[13]
Bladder cancer	EJ	S	[16]
Colon cance	HT-29	S, G2/M	[14]
Prostate cancer	22Rv1	G1	[17]
Prostate cancer	PC-3	S, G2/M	[17]
Liver cancer	SK-HEP-1, HCCLM3, Huh7	G2/M	[18,19]
Ovarian cancer	A2780, CP70	G2/M	[20]
Cholangiocarcinoma	HuCCT-1	G2/M	[21]
Renal cancer	786-O, 769-P	G2/M	[22]

KAE, kaempferol; GBC-SD, gallbladder carcinoma cell line; SGC996, gallbladder carcinoma cell line; EJ, bladder cancer cell line; HT-29, human colorectal adenocarcinoma cell line; 22Rv1, human prostate carcinoma cell line; PC-3, human prostate cancer cell line; SK-HEP-1, human liver adenocarcinoma cell line; HCCLM3, human hepatocellular carcinoma cell line; Huh7, human hepatocellular carcinoma cell line; A2780, human ovarian cancer cell line; CP70, cisplatin-resistant ovarian cancer cell line; HuCCT-1, human cholangiocarcinoma cell line; 786-O, human renal cell carcinoma cell line; 769-P, human renal cell carcinoma cell line; G0/G1, gap 0/gap 1 phase; S, synthesis phase; G2/M, gap 2/mitosis phase.

INDUCTION OF PROGRAMMED CELL DEATH IN TUMOR CELLS

Induction of apoptosis

While cell cycle arrest effectively suppresses the proliferation rate of tumor cells, the activation of cell death programs is essential for the complete eradication of the tumor. Building upon its ability to arrest the cell cycle, KAE further triggers various forms of programmed cell death.

1) Mitochondrial pathway

The mitochondrial pathway, also known as the intrinsic apoptotic pathway, is a critical mechanism for cells to execute programmed death in response to intracellular stress signals. This process is strictly regulated by the dynamic homeostasis of the Bcl-2 family proteins [23]. As a potent pro-oxidant, KAE triggers early apoptotic events in tumor cells, which are often accompanied by a burst of intracellular reactive oxygen species (ROS). The oxidative stress induced by KAE directly disrupts the balance within the Bcl-2 family, promoting the translocation of pro-apoptotic proteins (e.g., Bax) to the outer mitochondrial membrane and antagonizing anti-apoptotic proteins (e.g., Bcl-2). This leads to the opening of the mitochondrial permeability transition (MPTP) pore and the collapse of the mitochondrial membrane potential (ΔΨm). Subsequently, damaged mitochondria release pro-apoptotic factors, including cytochrome c, into the cytoplasm, inducing the formation of the apoptosome. This complex, in turn, activates the initiator caspase-9 and downstream effector caspases, ultimately executing the irreversible apoptotic program.

Previous studies have confirmed that KAE treatment significantly promotes the excessive accumulation of ROS in HepG2 liver cancer cells. This oxidative damage is a prerequisite for the decrease in mitochondrial membrane potential and the upregulation of the Bax/Bcl-2 ratio. The subsequent massive release of cytochrome c further triggers the caspase-9/3 cascade activation [24]. Additionally, recent research on head and neck cancer has demonstrated that KAE efficiently induces apoptosis by mediating mitochondria-dependent oxidative damage and synergistically inhibiting the activity of anti-apoptotic proteins [25]. However, it is worthwhile noting that these robust ROS bursts and subsequent apoptotic events are highly dose-dependent in vitro. Whether KAE can achieve sufficient concentrations to overcome the robust antioxidant defense systems within the complex in vivo tumor microenvironment remains a critical question requiring further investigation.

2) Death receptor pathway

The death receptor pathway, also known as the extrinsic apoptotic pathway, initiates the intracellular apoptotic cascade through the specific recognition and binding of extracellular lethal signals by members of the death receptor superfamily on the cell membrane. Lee et al. [26] have demonstrated that in colorectal cancer models, KAE treatment significantly upregulates the expression of membrane-bound FAS ligand. Concurrently, it induces the cleavage and activation of pro-Caspase-8 and full-length Bid (generating truncated Bid, tBid). The reduction in the levels of these pro-proteins directly reflects, and is accompanied by, a significant enhancement of caspase-8 enzymatic activity. Further in vitro analyses indicated that at a concentration of 60 μmoL/L, KAE significantly induces apoptosis in both HT-29 and p53-mutant SW480 human colorectal cancer cells.

Furthermore, in ovarian cancer, KAE directly upregulates the expression of death receptor 4 and death receptor 5 in human ovarian cancer cells (OVCAR-3 and SKOV-3) by activating the JNK/ERK-CHOP signaling pathway. This process effectively triggers the apoptotic program mediated by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [27].

3) Endoplasmic reticulum stress-mediated apoptosis

Endoplasmic reticulum stress (ERS) is a core regulatory mechanism for cells responding to the disruption of homeostasis. Studies have shown that KAE possesses significant ERS-inducing activity, leading to the pathological accumulation of unfolded or misfolded proteins within the endoplasmic reticulum (ER) lumen, which in turn triggers a persistent unfolded protein response (UPR). Recent compelling evidence has firmly established the critical role of the ERS pathway in KAE-induced apoptosis, particularly in colorectal cancer models [28].

The accumulated cytosolic Ca²+ acts as a central apoptotic signal through two primary mechanisms: first, it can be sequestered by the mitochondria, leading to calcium overload and the subsequent release of cytochrome c; second, it activates calcium-dependent proteases known as calpains. Activated calpains can directly cleave and activate caspase-12. This Ca²+-driven signal transduction network ultimately converges to initiate the caspase cascade, thoroughly executing the apoptotic program. These mechanisms are illustrated in Figure 2.

Figure 2. Molecular mechanisms of KAE-induced apoptosis through the extrinsic death receptor, intrinsic mitochondrial, and endoplasmic reticulum stress pathways.

The dashed lines indicate mechanistic crosstalk between different sub-pathways. KAE, kaempferol; ROS, reactive oxygen species; Cyt c, cytochrome; FasL, Fas ligand; DR4/5, death receptor 4/5; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; CHOP, C/EBP homologous protein; ERS, endoplasmic reticulum stress; XBP-1, X-box binding protein 1; PERK, protein kinase R-like ER kinase; tBid, truncated Bid; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2.

Specifically, in DLD-1 colorectal cancer cells, KAE disrupts ER homeostasis by upregulating the expression of XBP-1 and PERK, two key proteins in the UPR branching pathways. This process promotes the release of Ca²+ from the ER lumen into the cytoplasm [28], resulting in a significant elevation of cytosolic calcium levels.

Induction of autophagy

Under physiological conditions, autophagy primarily maintains cellular homeostasis. However, tumor cells often aberrantly upregulate it as metabolic fuel to support rapid proliferation [29]. In contrast to this protective mechanism, KAE can induce excessive, lethal autophagy by continuously enhancing autophagic flux. The inhibition of the PI3K/Akt/mTOR pathway is a primary mechanism for this effect. In cervical cancer and non-small cell lung cancer (NSCLC) cells, KAE relieves mTOR-mediated autophagic inhibition, leading to the massive formation of autophagic vacuoles and acidic vesicular organelles (AVOs) [30,31]. Conversely, KAE reactivates the AMPK pathway. In precancerous breast lesions, KAE-driven LKB1/AMPK reactivation promotes MFF/DRP1-mediated mitochondrial fission, triggering PINK1/Parkin-dependent lethal mitophagy to inhibit lesional tissue growth [32].

Importantly, the induction of autophagy by KAE is heavily intertwined with broad signaling networks that simultaneously govern apoptosis and cell cycle arrest, such as the MAPK cascade and ERS. KAE remodels MAPK signaling by downregulating p-ERK while activating JNK and p38 kinases, with JNK activation serving as a critical upstream trigger for autophagic flux [33]. Furthermore, intracellular calcium overload and subsequent ER stress represent highly interconnected nodes. In ovarian cancer cells, a KAE-induced Ca2+surge activates the UPR pathways, upregulating LC3-II and Beclin 1 while effectively suppressing pro-survival Akt signaling to reverse cisplatin resistance [34]. Similarly, in gastric cancer, KAE triggers autophagy via the cytoplasmic IRE1-JNK-CHOP axis, while uniquely acting as a histone deacetylase (HDAC) inhibitor to epigenetically drive lethal autophagy at the transcriptional level [35]. Ultimately, this excessive autophagic degradation disrupts cancer cell metabolic homeostasis, frequently converging with the mitochondrial apoptotic pathway to execute comprehensive cell death.

Induction of pyroptosis

Unlike non-inflammatory apoptosis, pyroptosis is a form of lytic programmed cell death accompanied by the release of pro-inflammatory cytokines. Its execution fundamentally depends on the formation of pore structures in the plasma membrane by the gasdermin family proteins. Recent studies have revealed that KAE can efficiently kill tumor cells and remodel the immune microenvironment by activating either canonical or non-canonical pyroptotic pathways, thereby overcoming the limitations of traditional apoptosis induction.

The pyroptosis executioner protein gasdermin E (GSDME) is regarded as a critical "molecular switch" that determines the mode of cell death. GSDME can be specifically cleaved by caspase-3 to release its N-terminal fragment, which possesses pore-forming activity, thereby shifting the initial apoptotic signal toward pro-inflammatory pyroptosis [36]. In glioma cells, KAE significantly elevates intracellular ROS levels and decreases mitochondrial membrane potential. High levels of ROS further induce autophagy, which serves as a prerequisite for the occurrence of pyroptosis; notably, treatment with the autophagy inhibitor 3-MA results in a concomitant reduction in the cleavage of GSDME [37].

Furthermore, in AGS gastric cancer cells, KAE tends to activate the canonical inflammasome pathway. This mechanism involves the activation of the NF-κB signaling pathway, which subsequently promotes the assembly of the NLRP3 inflammasome and triggers the self-cleavage of caspase-1. This sequence ultimately leads to the release of the mature pro-inflammatory cytokine IL-18 and cell lysis [38]. Importantly, by driving such pro-inflammatory lytic cell death, KAE actively contributes to the immunological remodeling of the tumor microenvironment, providing a strong mechanistic rationale for its prospective combination with ICIs.

Bidirectional regulation of ferroptosis

Ferroptosis is a unique form of regulated cell death characterized by the iron-dependent accumulation of lethal lipid peroxides [39]. Distinct from apoptosis, necrosis, and autophagy, the core driving force of ferroptosis lies in the functional failure of the glutathione (GSH)-dependent antioxidant defense system, which subsequently leads to uncontrolled lipid peroxidation of the plasma membrane [40].

Recent emerging evidence has robustly confirmed that KAE exerts potent anti-cancer activity by actively triggering the ferroptotic cell death program in malignant tumors. A comprehensive recent study demonstrated that KAE effectively induces ferroptosis in oral squamous cell carcinoma models, leading to a dose-dependent reduction in cancer cell viability [41]. Mechanistically, network pharmacology and molecular docking revealed that carbonic anhydrase acts as a core target gene. KAE exhibits a strong binding affinity for carbonic anhydrase IX, thereby severely disrupting cellular homeostasis and driving iron-dependent lipid peroxidation via the modulation of p53/FoxO signaling pathways [41].

Interestingly, KAE exhibits a remarkable cell-context-dependent bidirectional regulation of ferroptosis, contributing to its highly favorable safety profile. While it drives lethal lipid peroxidation in malignant cells, KAE acts as a potent antioxidant in normal tissues to prevent ferroptosis-induced injury. For instance, in normal human gastric epithelial cells (GES-1) exposed to oxidative stress, KAE effectively suppresses ferroptosis by strongly activating the Nrf2/glutathione peroxidase 4 (GPX4) antioxidant axis. It actively reduces intracellular ROS, malondialdehyde, and iron (Fe²+) accumulation while upregulating the protective SLC7A11 transporter [42]. This targeted selectivity—inducing ferroptosis to eradicate tumors while inhibiting ferroptosis to protect normal mucosa—highlights KAE's immense potential as an ideal therapeutic agent with minimal systemic toxicity.

Rather than acting on isolated pathways, KAE orchestrates a highly interconnected network of these non-canonical cell death modalities. To provide a comprehensive conceptual framework, the intricate mechanistic integration and crosstalk among KAE-induced apoptosis, autophagy, pyroptosis, and ferroptosis—particularly the central roles of ROS and ER stress, as well as the critical "apoptosis-to-pyroptosis switch"—are systematically summarized in Figure 3.

Figure 3. Mechanistic integration and crosstalk of KAE-induced non-canonical programmed cell death pathways.

Beyond classical apoptosis, KAE orchestrates a highly interconnected network of ferroptosis, autophagy, and pyroptosis. ROS accumulation and ER stress serve as central upstream nodes. The ROS surge directly inhibits the SLC7A11/GPX4 antioxidant axis, driving lethal lipid peroxidation and subsequent ferroptosis. Concurrently, ER stress activates the Beclin-1/LC3-II pathway to trigger excessive autophagic flux. Notably, this autophagy acts as a prerequisite for GSDME cleavage. The dashed line highlights a critical “Apoptosis-to-Pyroptosis Switch,” wherein activated caspase-3 specifically cleaves GSDME. The resulting GSDME-N terminal fragments oligomerize to form membrane pores, executing lytic pyroptosis and releasing pro-inflammatory cytokines (IL-18 and IL-1β). KAE, kaempferol; ROS, reactive oxygen species; ER, endoplasmic reticulum; SLC7A11, solute carrier family 7 member 11; GPX4, glutathione peroxidase 4; LC3-II, microtubule-associated protein 1A/1B-light chain 3; GSDME, gasdermin E; GSDME-N, gasdermin E N-terminal domain; IL-18, interleukin-18; IL-1β, interleukin-1 beta.

REGULATION OF THE TME

The TME represents a highly intricate ecosystem comprising malignant cells, recruited stroma, and an array of secreted factors, which collectively dictate tumorigenesis and malignant progression [43]. Malignant tumors can remodel host physiology by secreting signaling molecules, such as TNF and IL-6, which induce non-cell-autonomous autophagy within both the local microenvironment and distant tissues. Consequently, this tumor-driven stromal autophagy liberates essential nutrients—such as amino acids—providing vital metabolic support that fuels early tumor growth and subsequent invasion [44].

Inhibition of tumor angiogenesis

The neovascular network provides essential oxygen and nutrients for solid tumors. Angiogenesis within the tumor microenvironment is primarily driven by tumor-derived VEGF, which exerts mitogenic and anti-apoptotic effects, increases vascular permeability, and promotes cell migration. VEGF has been proven to play a central role in key signaling pathways mediating angiogenesis, as well as tumor growth and metastasis [45].

KAE exhibits broad-spectrum anti-angiogenic activity, primarily targeting the VEGF/VEGFR signaling axis. By inhibiting the ERK/NF-κB/c-Myc signaling pathway and inducing p21 expression, KAE ultimately downregulates VEGF levels, effectively blocking the blood supply required for tumor growth [46]. In hypoxic colorectal cancer cell models (HCT-15 and HCT-116), KAE not only inhibits VEGF expression driven by hypoxia-inducible factor-1α (HIF-1α) but also simultaneously blocks the Wnt/β-catenin signaling pathway and the epithelial-mesenchymal transition (EMT) process [47]. Importantly, conventional anti-angiogenic therapies frequently induce a severe hypoxic niche that paradoxically triggers EMT and subsequent metastasis. This dual capability of KAE—suppressing neovascularization while simultaneously halting hypoxia-driven EMT—highlights its translational potential to overcome the metastatic escape mechanisms often observed in current clinical practice.

Modulation of the TIME

The TIME, a vital component of the TME, is an immunosuppressive barrier formed by the interactive network of tumor cells, immune cells, and cytokines. It represents a major obstacle to clinical efficacy, contributing significantly to therapeutic resistance. Among the various immune populations, tumor-associated macrophages (TAMs) are the most abundant in the TIME. They predominantly exhibit an M2-like phenotype (characterized by high expression of CD206 and Arg-1), which actively promotes tumor growth, angiogenesis, and immune evasion.

Research has demonstrated that KAE exerts significant modulatory effects by reprogramming TAM polarization. On the one hand, KAE blocks the persistent activation of the STAT3 signaling pathway, leading to the downregulation of M2 phenotypic markers and the inhibition of the inflammatory chemokine CCL2 release. This action effectively curtails the recruitment of peripheral monocytes to the tumor bed and prevents their subsequent M2 polarization [48]. On the other hand, evidence suggests that KAE can enhance STAT1 phosphorylation, inducing a phenotypic repolarization of TAMs toward the classical M1 state, which exerts potent pro-inflammatory and tumoricidal functions [49].

By effectively reversing the M2-dominant immunosuppressive state into an immune-active TIME, KAE facilitates the reinvigoration of the host's anti-tumor cellular immunity. This profound immunological remodeling has critical translational implications. The conversion of an immunosuppressive "cold" tumor into an inflamed "hot" tumor provides a compelling mechanistic rationale for the synergistic application of KAE with ICIs, such as anti-PD-1 or anti-PD-L1 therapies. Preconditioning the TIME with KAE to relieve macrophage-mediated T-cell suppression could potentially sensitize refractory tumors to immunotherapy, representing a highly promising avenue for future clinical trials.

Inhibition of extracellular matrix (ECM) degradation

The remodeling of the ECM is a prerequisite for tumor invasion and distant metastasis, with matrix metalloproteinases (MMPs) playing a pivotal role in ECM degradation. KAE exhibits significant anti-invasive activity by effectively blocking MMP-mediated matrix degradation.

Research indicates that in hepatocellular carcinoma, KAE inhibits the phosphorylation of the Akt signaling pathway, thereby downregulating the protein expression and enzymatic activity of MMP-9. This action subsequently weakens the ability of cancer cells to penetrate the basement membrane [50]. Furthermore, in tongue squamous cell carcinoma models, KAE inhibits the expression of MMP-2 and its tissue inhibitor, TIMP-2, at the transcriptional level by blocking the ERK/c-Jun signaling axis [51]. Collectively, KAE can target upstream regulators such as Akt or ERK/c-Jun across various tumor contexts to inhibit MMP-2/9 activity through multiple pathways, thereby maintaining ECM integrity and curbing the malignant progression of tumors. Given that numerous synthetic MMP inhibitors have historically failed in clinical trials due to severe musculoskeletal toxicities and lack of specificity, the ability of KAE to naturally and indirectly modulate MMP activity via upstream signaling axes suggests it may offer a safer, multi-targeted alternative for anti-metastatic intervention.

COMBINATION THERAPY AND REVERSAL OF CHEMORESISTANCE

Current anti-tumor clinical practice has shifted from monotherapy to a multidisciplinary integrated treatment model, including surgical resection, radiotherapy, chemotherapy, molecular targeted intervention, and immune checkpoint blockade. However, surgery and radiotherapy are primarily effective for localized lesions and struggle to eradicate micro-residual disease or distant metastases. While systemic chemotherapy can target tumor cells throughout the body, it is frequently associated with severe bone marrow suppression, neurotoxicity, and multidrug resistance, leading to treatment failure, patient intolerance, and a high incidence of adverse effects that severely impair the quality of life [52]. Notably, the combination of KAE with conventional therapeutic agents offers a promising strategy to significantly alleviate these pronounced side effects while conquering robust drug resistance.

Synergistic sensitization and toxicity mitigation

When combined with classical chemotherapeutics, KAE functions as a potent sensitizer, amplifying the lethal effects of the drugs through complementary signaling networks. For instance, docetaxel is a first-line chemotherapeutic agent for advanced prostate cancer; its combined use with KAE has been shown to simultaneously induce profound autophagy and apoptosis in prostate cancer cells, achieving superior efficacy at lower, less toxic doses [53]. Similarly, when combined with doxorubicin, KAE exhibits significant synergistic anti-tumor potential by downregulating cyclin D1 to inhibit cell proliferation, suppressing anti-apoptotic Bcl-2, and modulating the autophagic process via Beclin-1 [54]. Furthermore, in HCT-8 and HCT-116 cells, the combination of KAE and 5-fluorouracil (5-FU) was found to markedly inhibit the activation of the PI3K/Akt pathway and downregulate thymidylate synthase, thereby maximizing apoptotic induction [55].

Overcoming multidrug resistance

Chemoresistance remains the primary cause of treatment failure, and KAE has been shown to reverse this resistance by targeting the fundamental metabolic and phenotypic vulnerabilities of refractory cells. One critical mechanism involves metabolic reprogramming. In colorectal cancer cells, KAE promotes the expression of microRNA-326 (miR-326), which directly targets the 3′-UTR of the pyruvate kinase M2 (PKM2) isoform to inhibit glycolysis. By suppressing PKM2-driven glycolytic metabolism, KAE successfully reverses the acquired resistance of colorectal cancer cells to 5-FU [56].

Additionally, KAE reverses platinum-based resistance through multi-target intervention. It produces a significant synergistic effect with cisplatin, enhancing cytotoxicity against resistant colorectal cancer cells by shifting the Bax/Bcl-2 balance and enforcing cell cycle arrest [57]. Targeting the ribosomal S6 kinase with KAE has also been identified as an effective alternative strategy to re-sensitize oxaliplatin-resistant colorectal cancer models [58]. Finally, KAE directly dismantles tumor stemness, a root cause of MDR. Research in breast cancer demonstrated that KAE, particularly when used alongside MDR modulators like verapamil, exerts a profound chemosensitizing effect by downregulating core stemness transcription factors (such as SOX2 and OCT4) and disrupting the CD44-NANOG-MDR1 resistance complex [59].

CROSSTALK BETWEEN KAE AND THE GUT MICROBIOME

Acting as the critical interface between dietary intake and systemic physiology, the gut microbiome profoundly influences the initiation, progression, and prognosis of malignancies [60]. Its composition and dynamic alterations also serve as critical determinants of patient responses to systemic therapies, including chemotherapy and immunotherapy. Consequently, targeted modulation of the gut microbiota has emerged as a pivotal strategy for optimizing anti-tumor efficacy, mitigating treatment-induced toxicities, and innovating cancer intervention paradigms [61].

Within this context, the interplay between KAE and the gut microbiota represents a novel and highly promising frontier in the field of cancer prevention. This interaction is inherently bidirectional. On the one hand, specific intestinal microbial taxa can enzymatically cleave KAE into smaller, highly bioavailable phenolic acid derivatives, thereby facilitating its systemic absorption. Conversely, KAE exerts a remarkable prebiotic-like effect by remodeling the architectural composition of the gut microbiota. A compelling in vivo study utilizing a Lewis lung carcinoma mouse model provides direct evidence for this mechanism [62]. The researchers demonstrated that KAE administration not only significantly suppressed tumor growth but also selectively enriched the abundance of beneficial microbial taxa, particularly Lactobacillus, Lachnospiraceae, and Bacteroides (e.g., B. acidifaciens). Crucially, this microbial reconfiguration was found to be highly correlated with the systemic activation of cytotoxic T lymphocytes and natural killer (NK) cells.

This microbiome-mediated immunostimulation is further bolstered by the enrichment of bacterial metabolites, such as short-chain fatty acids, which are known to mitigate mucosal inflammation and prime systemic immune responses. Consequently, by restoring intestinal microbial homeostasis, KAE not only impedes carcinogenesis but also actively orchestrates a "hot" (i.e., immune-inflamed and highly infiltrated) tumor microenvironment. Given its capacity to activate cytotoxic T cells via the gut-immune axis, KAE exhibits immense, yet largely untapped, synergistic potential when administered in combination with ICIs. Moving forward, rigorous clinical trials are urgently warranted to comprehensively elucidate this microbiome-dependent antineoplastic axis and to translate these synergistic strategies into clinical oncological practice.

OTHER PHARMACOLOGICAL ACTIVITIES

Beyond its prominent direct anti-tumor and chemosensitizing effects, KAE exhibits a pleiotropic pharmacological profile characterized by exceptional antioxidant and anti-inflammatory activities. In terms of antioxidant capacity, KAE possesses potent free radical scavenging capabilities due to the multiple phenolic hydroxyl groups within its molecular structure. By activating the Nrf2/HO-1 antioxidant signaling pathway [63] and upregulating the activities of endogenous enzymes such as superoxide dismutase (SOD) and GPX, KAE effectively alleviates intracellular oxidative stress, protects genomic stability, and blocks the malignant transformation of normal cells at the initiation stage.

Concurrently, KAE demonstrates robust anti-inflammatory activity by inhibiting the overactivation of key inflammatory cascades, such as the NF-κB and MAPK pathways, thereby downregulating the expression of pro-inflammatory mediators including TNF-α, IL-6, COX-2, and iNOS. Crucially, these broad-spectrum protective effects endow KAE with tremendous potential to mitigate the severe systemic toxicities frequently induced by conventional chemotherapy. For instance, KAE can readily cross the blood-brain barrier (BBB) to exert neuroprotective effects [64], offering a prospective strategy to alleviate chemotherapy-induced peripheral neuropathy (CIPN). Similarly, the renoprotective effects of KAE have been robustly substantiated by clinical epidemiological data: in overweight populations, dietary KAE intake is significantly and negatively correlated with the risk of renal injury, with every 5 mg increase in daily dosage yielding a 7% reduction in disease risk [65]. This highlights its potential to counteract nephrotoxic drugs like cisplatin.

Additionally, recent breakthroughs have elucidated the role of KAE in regulating gastrointestinal motility. By directly acting on L-type calcium channels in smooth muscle cells via a non-neuronal dependent regulatory mode, it inhibits colonic contraction [66], which could be highly beneficial for managing chemotherapy-induced gastrointestinal dysfunction. As research deepens, the pleiotropy demonstrated by KAE indicates that its therapeutic value in bridging oncological interventions with systemic organ protection warrants extensive clinical exploration.

CONCLUSION AND FUTURE PERSPECTIVES

An extensive literature review reveals that the anti-tumor effects of KAE on a single tumor type are pleiotropic rather than isolated, involving complex crosstalk between various mechanisms (e.g., the synergistic induction of autophagy and ferroptosis). While it is well-established that KAE modulates classical signaling cascades such as PI3K/Akt, MAPK, and NF-κB [67], current research predominantly focuses on the phenotypic validation of downstream effects. There remains a glaring lack of rigorous target identification studies concerning the direct molecular targets of KAE and its physical interaction patterns upon entering the host cells.

Despite the extensive preclinical data supporting KAE’s multifaceted anticancer efficacy, current research exhibits significant experimental limitations that must be critically acknowledged. Firstly, a substantial translational gap exists due to the reliance on in vitro models utilizing supraphysiological concentrations (e.g., 20-80 μM), which do not accurately reflect the physiologically achievable systemic levels in humans following conventional oral administration. Secondly, there is a noticeable "cell line bias"; the majority of studies depend on highly homogenous, established commercial cell lines cultured in 2D monolayers, which inherently fail to recapitulate the complex spatial architecture and heterogeneous TME of actual human solid tumors. Furthermore, while current literature frequently highlights multiple downstream targets based on preliminary in vitro phenotypic assays or in silico computational predictions, rigorous in vivo target engagement validations—such as genetic knockout models—remain markedly sparse. Consequently, there is a severe paucity of human clinical trial data to validate the actual pharmacokinetic (profile of KAE and establish physiologically effective therapeutic doses in patients.

The clinical translation of KAE is primarily hindered by its suboptimal pharmacokinetic characteristics, including poor aqueous solubility, rapid metabolic clearance, and consequent low bioavailability. The rapid evolution of nanotechnology and innovative delivery systems has offered viable solutions to optimize its tissue uptake and biodistribution [68]. For instance, a D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-stabilized KAE nanosuspension [69] has demonstrated significantly enhanced stability, solubility, and anti-tumor efficacy. However, despite the pharmacokinetic advantages of these formulations, a critical oversight in current research is the absence of comprehensive, long-term toxicological profiling. Issues such as the potential accumulation toxicity of non-biodegradable polymeric materials in the reticuloendothelial system, alongside potential immunogenicity, pose major hurdles. Future pharmaceutical endeavors must not only focus on improving targeted delivery but also rigorously address the long-term biosafety and clinical feasibility of these nano-carriers to bridge the gap from bench to bedside. Ultimately, integrating these advanced delivery platforms with novel therapeutic paradigms—such as microbiome-targeted interventions and immune checkpoint blockade—will unlock the full translational potential of KAE in precision oncological practice.