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Review

Hesperidin and Hesperetin: Epigenetic-Stemness Crosstalk, Antitumor Mechanisms, Preclinical Data and Translation Barriers

by
Mengqi Guo
,
Linxin Shao
,
Huiqing Yin
,
Qianrui Kou
,
Lele Shang
,
Haixia Guan
and
Fang Li
*
Yan’an Medical College, Yan’an University, Yan’an 716000, China
*
Author to whom correspondence should be addressed.
Biomolecules 2026, 16(7), 1063; https://doi.org/10.3390/biom16071063
Submission received: 15 May 2026 / Revised: 25 June 2026 / Accepted: 26 June 2026 / Published: 21 July 2026
(This article belongs to the Section Natural and Bio-derived Molecules)
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Abstract

Hesperidin is a natural flavonoid derived from citrus plants, which can be hydrolyzed into hesperetin in vivo. Both compounds have anti-inflammatory, antioxidant and antitumor activities. At present, there is a lack of reviews focusing on the epigenetic regulation of cancer stem cells (CSCs) mediated by hesperidin and hesperetin. This review summarizes the molecular crosstalk between hesperidin/hesperetin and CSCs mediated via three major epigenetic pathways, including direct regulatory effects, indirect modulatory actions, and mechanistic relationships proposed based on scientific hypotheses. We elaborate their effects on inhibiting the self-renewal, invasion and metastasis of CSCs as well as reversing chemoresistance, and analyze the crosstalk between epigenetic networks and classical signaling pathways of CSCs. Furthermore, we discuss the core bottlenecks restricting the clinical transformation of these two compounds and introduce improvement strategies such as nanodelivery systems. Current research is still confronted with problems including CSC heterogeneity and the potential off-target toxicity of drugs. In conclusion, hesperidin and hesperetin may serve as potential candidate agents for epigenetic regulation targeting CSCs, which can offer novel theoretical basis for comprehensive tumor therapy.

1. Introduction

1.1. Research Potential of Natural Compounds in Tumor Therapy

The continuously increasing global incidence and mortality of cancer make it urgent to explore the pathogenesis of cancer and identify new therapeutic targets and drugs [1]. Natural flavonoids have attracted extensive attention in tumor research due to their good biocompatibility and multi-target regulatory effects [2]. Hesperidin, a typical flavonoid widely distributed in citrus plants, exerts anticancer, anti-inflammatory and antioxidant effects. Hesperetin can be generated from hesperidin via hydrolysis catalyzed by intestinal rhamnosidase, and it can also be extracted by industrial methods. This transformation process may serve as an important basis for its antitumor activity [3,4]. Up to now, preliminary antitumor studies of hesperidin and hesperetin have been carried out in various tumor models such as ovarian cancer, breast cancer and leukemia, showing favorable application potential [5,6,7].

1.2. Roles of Epigenetic Regulation in Tumor Oncogenesis and Progression

Epigenetic regulation refers to the process of modulating gene expression via DNA methylation, histone modification, non-coding RNA regulation, chromatin remodeling and other approaches without altering DNA sequences [8]. DNA methylation mainly inhibits gene expression by adding methyl groups to CpG islands, and recruits inhibitory methyl-binding proteins to maintain gene silencing [9]. Accumulating studies have demonstrated that aberrant DNA methylation plays a key regulatory role in glioblastoma, gastric cancer and breast cancer [10].
Histone modification refers to post-translational modifications (PTMs) of N-terminal amino acids of histones, including histone methylation, histone acetylation and histone ubiquitination [11]. Histone modification is closely associated with lung cancer, breast cancer, head and neck squamous cell carcinoma and colorectal cancer [12,13,14,15]. Moreover, microRNAs (miRNAs), a type of non-coding RNA, may exert critical functions in the regulation of tumor gene expression [16].

1.3. Roles of Cancer Stem Cells in Tumor Oncogenesis and Progression

Cancer stem cells (CSCs) are a cell population with strong self-renewal ability, cellular plasticity and drug resistance, which are key drivers of tumor development and chemoresistance [17]. CSCs can differentiate into multiple tumor cell subtypes, so as to maintain and regulate tumor heterogeneity [18]. Classic CSC biomarkers include CD133, CD44 and ALDH1 [19,20]. CD133 is widely expressed in colorectal cancer, lung cancer, prostate cancer and bladder cancer [21,22,23]. CD44 is commonly detected in liver cancer, breast cancer and head and neck squamous cell carcinoma [24,25,26]. ALDH1 is also recognized as a biomarker of breast cancer. Elevated ALDH1 expression is correlated with HER2 positivity and is often associated with poor prognosis [27]. Besides breast cancer, ALDH1 activity is also widely utilized as a biomarker for CSCs in other solid tumors, including head and neck squamous cell carcinoma, colorectal cancer and lung cancer [28,29,30].
In addition, extensive aberrant DNA methylation exists in CSCs, leading to the silencing of tumor suppressor genes and activation of CSC-related genes [31]. Epigenetic modifications and CSCs may cooperatively modulate tumor growth, metastasis, and chemoresistance [32]. Based on the above background, this review centers on hesperidin and hesperetin, and elaborates their molecular crosstalk with CSCs through epigenetic modifications, alongside their pharmacological properties and obstacles in clinical translation, aiming to lay a foundation for fundamental research and translational development of these natural flavonoids.

2. Regulatory Effects of Hesperidin and Hesperetin on Tumor Growth, Metastasis and Chemoresistance

Benefiting from the characteristic of multi-target regulation, hesperidin shows great potential in inhibiting tumor growth and metastasis and alleviating tumor cell chemoresistance. In the group treated with hesperidin (10 μmol/L) combined with paclitaxel (PTX, 10 μmol/L), the cell viability was 16.38%, the proportion of apoptotic cells reached 83.68%, and the number of migratory cells decreased by 65%, which was significantly different from the group treated with PTX (20 μmol/L) alone. The tumor inhibition rate of combined treatment with hesperidin and PTX was 66.17%. Hesperidin may induce apoptosis of oral squamous cell carcinoma (OSCC) cells by upregulating the expression of Bax and caspase-3. Meanwhile, it significantly downregulates MMP-9 and N-cadherin, key molecules involved in EMT, and upregulates E-cadherin, thereby reducing the migration ability of OSCC cells [33].
In vitro combined treatment with hesperetin (200, 400, 600, 800 μM) and 5-fluorouracil (5-FU, 20, 40, 60, 80 μM) as well as in vivo intervention with hesperetin (60 mg/kg) combined with 5-FU (10 mg/kg) achieved better antitumor effects than monotherapy. The combination arrested esophageal cancer cells at the G0/G1 phase, inhibits the PI3K/AKT signaling pathway, upregulates p21, and downregulates Cyclin D1, MMP-2 and MMP-9, thereby hindering the proliferation and invasion of esophageal cancer cells (Eca-109). Hesperetin combined with 5-FU activated the mitochondrial apoptotic pathway, reduced the Bcl-2/Bax ratio, and triggered the activation of caspase-3 and caspase-9 to induce the apoptosis of esophageal cancer cells [34].
In the Ehrlich ascites carcinoma (EAC) mouse model, combined administration of hesperidin (100 mg/kg) and cisplatin (5 mg/kg) exerted synergistic anticancer effects, which were superior to single-drug treatment. The combination further downregulated the tumor proliferation marker Ki-67 and upregulated the apoptotic effector protein caspase-3, enhancing the effects of cisplatin on inhibiting tumor proliferation and inducing cell apoptosis [35]. After treatment with 100 μM and 200 μM hesperidin alone, the cell viability decreased by 45.1% and 65.2% respectively. After treatment with 25 μM and 50 μM cisplatin alone, the cell viability decreased by 37.4% and 48.4% respectively. Compared with cisplatin monotherapy and hesperidin monotherapy, the cell viability of malignant melanoma cells (A431) was reduced to 21.7% ± 2.8% in the group treated with 108.4 μM hesperidin combined with 32.8 μM cisplatin. The two agents synergistically upregulated the expression and protein activity of pro-apoptotic genes Bax, caspase-3 and caspase-7, and significantly downregulated the anti-apoptotic gene Survivin, which greatly increased the apoptosis rate of malignant melanoma cells and enhanced cytotoxicity [36].
Combined treatment with hesperidin (25 μM) and cisplatin (25 μM) significantly enhanced the inhibitory effect on laryngeal carcinoma Hep-2 cells by regulating the TRPM2 channel compared with monotherapy. Activated TRPM2 mediated massive extracellular Ca2+ influx and disrupted cellular calcium homeostasis. Meanwhile, it induced abnormal accumulation of ROS, depletion of glutathione (GSH) and elevation of malondialdehyde (MDA), aggravating cellular oxidative stress and lipid peroxidation damage. The mitochondrial membrane potential (ΔΨm) decreased, leading to mitochondrial dysfunction. In addition, the combination of hesperidin and cisplatin upregulated the expression of pro-inflammatory cytokines IL-1β and TNF-α, and ultimately induced apoptosis of human laryngeal squamous cell carcinoma Hep-2 cells, effectively enhancing the in vitro antitumor cytotoxicity of cisplatin [37].
Combined treatment with hesperidin (50–200 μM) and gemcitabine (10–50 μM) exerted synergistic anti-proliferative effects on human endometrial carcinoma cells ISHIKAWA. The most prominent effect was observed in the group treated with hesperidin (100 μM) plus gemcitabine (10 μM), with a tumor inhibition rate of 38–42% at 24 h and 26–30% at 48 h. The related mechanisms are as follows: Firstly, hesperidin combined with gemcitabine induced cell apoptosis by regulating the mitochondrial apoptotic pathway, including upregulating pro-apoptotic protein Bax, downregulating anti-apoptotic protein Bcl-2, and activating caspase-3/7 protease activity. Meanwhile, it downregulated HIF-1α and VEGF expression to block tumor hypoxia adaptation and angiogenesis. In terms of redox homeostasis, hesperidin neutralized excessive reactive oxygen species (ROS) induced by gemcitabine, realizing dual effects of chemotherapy sensitization and toxicity reduction [38].
Compared with monotherapy, combined treatment with hesperidin (10–1000 μM) and doxorubicin (10–1000 nM) significantly increased caspase-3 level; decreased Bcl-2, IL-1β, IL-6 and TNF-α levels; and promoted apoptosis of HeLa cervical cancer cells [39]. Furthermore, compared with sorafenib monotherapy, combined treatment with 1.5 mmol/L hesperidin and 1.0 μmol/L sorafenib remarkably reduced the expression of CSC markers (CD44, CD133) and β-catenin in hepatocellular carcinoma cells, and significantly increased caspase-3 expression, thereby strengthening the inhibitory effect on proliferation and pro-apoptotic effect of HepG2 cells [40].
Collectively, in vitro and in vivo experiments have verified that the combination of hesperidin or hesperetin with platinum drugs, taxanes, fluorouracil, gemcitabine and other chemotherapeutic agents can exert synergistic antitumor effects through multiple mechanisms, including activating the mitochondrial apoptotic pathway, inhibiting EMT, eliminating excessive ROS and blocking angiogenesis. Certain combination therapeutic regimens can also alleviate the oxidative damage induced by chemotherapeutic agents, thereby exerting synergistic antitumor effects (Table 1).

3. Key Signaling Pathways and Molecular Targets of Hesperidin and Hesperetin in Regulating Cancer Stem Cells and Epigenetics

3.1. Molecular Mechanisms of Hesperidin and Hesperetin in Tumor Epigenetic Modification

Epigenetic modification plays an important role in tumor development and progression, and the regulation of epigenetics by natural drugs has become a hot research topic in recent years [41].

3.1.1. Direct Regulation

Studies have confirmed that treatment of gastric cancer cells (MKN45, HGC27) with hesperetin (≤100 μM) downregulated the expression of histone acetyltransferase CBP, reduced DOT1L acetylation and degradation, and further decreased H3K79 methylation. This process downregulated CDH2, FN1, Twist, MMP2 and MMP9, and upregulated CDH1, ultimately inhibiting the migration and invasion of gastric cancer cells [42].
MicroRNAs (miRNAs) are important post-transcriptional epigenetic regulators in tumor development [43]. MiR-21-5p and miR-155-5p are abnormally highly expressed in breast cancer, which may accelerate malignant progression by inhibiting tumor cell apoptosis and promoting cell cycle progression, and are considered potential epigenetic therapeutic targets for breast cancer [44]. After breast cancer cells (MCF-7) were intervened with hesperidin nanoemulsion (29.36 μg/mL, 100 μg/mL) for 24 h, the transcriptional levels of miR-21 and miR-155 were significantly downregulated, arresting the cell cycle at the G2/M phase and further promoting the apoptosis of the breast cancer cells [45].
Other researchers treated breast cancer cells (MDA-MB-231) with hesperetin (10–500 μM). The results showed that there was a unique interaction between miR-486-5p and lncRNA H19, and the two presented an obvious bidirectional regulatory relationship. Hesperetin may upregulate miR-486-5p to directly downregulate lncRNA H19 and ICAM-1 expression. It can also inhibit the expression of downstream ICAM-1 by downregulating lncRNA H19, and ultimately suppress the proliferation, colony formation and migration of breast cancer cells [46].

3.1.2. Indirect Regulation

Treatment of breast cancer cells (BT-474, SKBR3) with hesperetin (200, 400, 600 μM) significantly upregulated the expression of MLH1 and MSH2. As core genes of the DNA mismatch repair system, the upregulation of MLH1 and MSH2 is obviously associated with the promotion of breast cancer cell apoptosis [47,48]. Studies have shown that tumor cells and cisplatin-resistant cell lines exhibit hypermethylation in the MLH1 promoter region, revealing a close correlation between MLH1 and epigenetics [49,50]. Hesperetin may activate the mismatch repair pathway to induce tumor cell apoptosis via epigenetic approaches, which remains to be verified by further experiments.
Treatment of human B lymphoblastic leukemia cells (NALM-6) with hesperidin (10–100 μM) for 24 h upregulated PPARγ. On the one hand, it increased the expression of p53 and p21 to induce cell cycle arrest and initiate apoptosis. On the other hand, it inhibited IκB phosphorylation and suppressed NF-κB activity to promote tumor cell apoptosis. Meanwhile, hesperidin upregulated Bax, downregulated Bcl-2 and XIAP, and activated caspase-3/9 within 24 h, jointly inducing apoptosis of NALM-6 cells [51]. It has been reported that PPARγ is a target of epigenetic modification and plays an important role in tumor regulation [52]. Therefore, hesperidin may exert antitumor effects via epigenetically regulating PPARγ (Figure 1).

3.2. Molecular Mechanisms of Hesperidin and Hesperetin in Regulating CSCs

CSC-related transcription factors including OCT4, SOX2, NANOG, MYC, CCND1, LGR5 and CXCR-4 integrate multiple signaling pathways such as Wnt/β-catenin, Notch, NF-κB, TGF-β/SMAD and JAK-STAT to form a molecular regulatory network, thereby regulating CSC stemness [53,54,55,56].

3.2.1. Direct Regulation

Firstly, hesperidin may regulate transcription factors and signaling pathways to inhibit CSCs. Treatment of breast cancer cells (MDA-MB-231) with hesperidin (12.5–100 μM) stabilized the G-quadruplex silencing element Pu-27 in the c-Myc promoter region, inhibited c-Myc transcription and downregulated the expression of downstream CCND1, which may block the proliferation and impair the self-renewal ability of breast CSCs [57].
Treatment of non-small cell lung cancer cells (A549, H460, H1975) with hesperidin (25–62.5 μg/mL) inhibited SDF-1α secretion and downregulated CXCR-4 to block the activation of the SDF-1/CXCR-4 signaling axis. This process reduced the expression of NF-κB (p-p65, p-IκB) and PI3K/Akt (p-Akt), further downregulated Vimentin and MMP-9, upregulated CK-19, reversed EMT, and inhibited the migration and invasion of lung cancer cells [58].
Studies have demonstrated that the self-renewal, proliferation and drug resistance of CSCs are mainly regulated by the classical Wnt and Notch signaling pathways [59,60]. In vitro treatment with 0.5 mmol/L and 1.5 mmol/L hesperidin and in vivo administration at 400 mg/kg blocked the Wnt pathway and inhibited the nuclear translocation of β-catenin, thereby downregulating CD44 and CD133. Meanwhile, it upregulated Bax and caspase-3 and downregulated Bcl-2, ultimately inducing apoptosis of hepatocellular carcinoma cells [40].
Treatment of breast cancer MCF-7 cells with hesperidin (100 μM, 200 μM) inhibited the PI3K/Akt pathway, downregulated MDM2 and upregulated p53 expression. Further regulation by p53 includes inducing G0/G1 cell cycle arrest to inhibit cell proliferation, activating apoptotic signals to promote cell death, downregulating ALDH1 to suppress breast CSC stemness, spheroid formation and colony formation, and downregulating MMP9 to inhibit cell migration and invasion. Combined with bioinformatics analysis, p53 was identified as the core target of hesperidin acting on breast CSCs, and the targeted regulatory mechanism between them needs further in-depth research [61].

3.2.2. Indirect Regulation

Treatment of lung squamous cell carcinoma cells with hesperetin (H1703: 37.5–150 μM, H226: 75–300 μM) inhibited the Notch1 signaling pathway and activated endoplasmic reticulum stress-related proteins p-eIf2α, CHOP and Grp78. After the treatment of hesperetin, tumor cells were arrested at the G2/M phase, accompanied by the upregulation of pro-apoptotic protein Bax and Cleaved-Caspase-3, downregulation of Cyclin B and CKD1, and decreased mitochondrial membrane potential, which ultimately induced apoptosis of lung squamous cell carcinoma cells [62]. Given that Notch1 can promote the nuclear translocation of β-catenin, the dual inhibition of Wnt/β-catenin and Notch pathways by hesperidin blocks the crosstalk between the two signaling pathways, which may serve as one of the potential mechanisms underlying its suppressive activity against CSCs [63] (Figure 2).

3.3. Crosstalk Between Epigenetic Modification and CSCs: Mechanisms Underlying the Effects of Hesperidin and Hesperetin

Extensive aberrant DNA methylation exists in CSCs, and the inhibition of DNA methyltransferases (DNMTs) can reduce the CSC pool and suppress tumorigenesis [64,65]. Histone acetylation and methylation can remodel chromatin structure and affect the transcriptional activity of CSC-related genes [66]. In addition, epigenetic regulatory mechanisms such as DNA methylation, non-coding RNA and histone modification may interact with CSC-related signaling pathways including Wnt/β-catenin, Notch and Hedgehog [67,68,69].

3.3.1. Direct Regulation

Treatment of non-small cell lung cancer cells (A549, H460) and xenograft tumors with hesperidin (1 μM, 2.5 μM in vitro; 60 mg/kg in vivo) upregulated miR-132, which directly targeted and silenced the CSC-related gene ZEB2, thereby exerting anti-proliferative and pro-apoptotic effects [70,71]. The above findings provide references for the study on the regulation of CSCs by hesperidin via epigenetics.

3.3.2. Indirect Regulation

Studies have shown that treatment of hepatocellular carcinoma cells (HepG2) in vitro and intervention in rat liver cancer models in vivo with hesperidin (50–400 μM in vitro; 150 mg/kg in vivo) inhibited both canonical Wnt3a/β-catenin and non-canonical Wnt5a pathways, downregulated β-catenin and its downstream Cyclin D1, upregulated Caspase-3, and induced the apoptosis of hepatocellular carcinoma cells [72]. Hypermethylation of the Wnt5a promoter region has been reported during tumor development, and Wnt5a plays an essential role in sustaining the survival and stemness of CSCs [59,73]. However, it remains to be elucidated whether hesperidin regulates CSCs via epigenetic modification of Wnt5a, which requires further comprehensive experimental verification.

3.3.3. Putative Mechanistic Hypothesis

Hesperetin (10–100 μg/mL) binds to TOP2A protein and significantly downregulates TOP2A expression in lung cancer cells (A549), thereby blocking DNA replication and cell proliferation, accompanied by mitochondrial dysfunction and eventual cell apoptosis [74]. TOP2A has been reported to interact with epigenetic regulators such as BAZ2A, KDM1A and EZH2, and to be modulated by RNA epigenetic modifications, particularly m6A methylation [75,76,77]. In non-tumor models, hesperidin (0.25 μM) was shown to downregulate m6A expression in human villous trophoblasts under inflammatory/high-glucose conditions [78]; however, whether hesperidin exerts similar effects on m6A in cancer cells remains unknown. Regarding epigenetic regulation of TOP2A, norcantharidin has been demonstrated to inhibit H3K27me3, which may in turn suppress TOP2A expression and upregulate p53, leading to apoptosis in hepatocellular carcinoma cells [79]. This suggests that epigenetic modifications can regulate TOP2A, but no study has yet reported that hesperidin or hesperetin can regulate TOP2A through epigenetic mechanisms. In prostate cancer, TOP2A negativity is considered a feature of CSCs and is associated with drug resistance [80]. These observations indicate that TOP2A may serve as a potential link between epigenetic modifications and CSC characteristics. Given that hesperidin can modulate both TOP2A expression and m6A levels (in different contexts), it is plausible that hesperidin could influence the epigenetic-TOP2A-CSCs axis, although direct experimental evidence in tumor models is currently lacking [81].
Hesperidin (10–400 μM) downregulated FOXP3 expression in ovarian cancer cells (SKOV3) via two parallel pathways: Pathway 1: hesperidin upregulated Bax and Caspase-3 to activate the endogenous apoptotic pathway and directly induce apoptosis of SKOV3 cells. Pathway 2: hesperidin downregulated FOXP3 and TNF-α while upregulating IFN-γ, relieving immunosuppression in the tumor microenvironment and remodeling antitumor immunity [82]. Studies have indicated that the induction and maintenance of FOXP3 are accompanied by epigenetic modifications such as histone acetylation and DNA demethylation [83]. In addition, FOXP3 can bind to p65 and inhibit the transcription of the stemness factor COX2 mediated by NF-κB, thereby suppressing CSCs and the malignant progression of colorectal cancer [84]. Therefore, the expression of FOXP3 may be regulated by epigenetic modifications including DNA methylation and histone acetylation; meanwhile, FOXP3 is capable of modulating stemness-related pathways in CSCs. However, whether the regulation of FOXP3 by hesperidin mediates the crosstalk between epigenetics and CSCs remains to be verified by experiments (Figure 3).

4. Preclinical Research on Hesperidin: Challenges and Optimization Strategies

4.1. Optimization of Drug Delivery Systems: Addressing the Bottleneck of Low Bioavailability

The main obstacle restricting the clinical transformation of hesperidin is its low lipophilicity, which leads to insufficient bioavailability and impairs its absorption and distribution in vivo [85]. Studies have shown that after healthy volunteers consumed orange juice, hesperidin was hydrolyzed into hesperetin in the intestinal tract. The peak plasma concentration of hesperetin was extremely low, only 0.46 μM and 1.28 μM after intake of 0.5 L and 1 L orange juice respectively. Concentrations commonly applied in current in vitro antitumor experiments fall within the range (50 μM–200 μM), yet direct oral administration of hesperidin or hesperetin monomers to human subjects has not been conducted in relevant studies [86]. Thus, extensive clinical trials are still needed to characterize the peak plasma concentrations of orally administered hesperidin and hesperetin monomers in humans.
Hesperidin methyl chalcone (HMC) was synthesized via methylation to alleviate acute renal injury in rats. When administered via intraperitoneal injection, HMC exerted dose-dependent protective effects within the dose range of 0.03–3 mg/kg, and 3 mg/kg was the optimal effective dose. It significantly activated the Nrf2 pathway, inhibited oxidative stress and inflammation, and reversed renal function damage. HMC remarkably improved the lipophilicity and bioavailability of hesperidin, but its antitumor activity has not been verified so far, and experiments are still needed for further exploration [87].
In prostate cancer research, hesperidin and berberine were co-encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The average particle size of PLGA nanoparticles was less than 200 nm with a polydispersity index lower than 0.1. The hesperidin-loaded PLGA nanoparticles presented a spherical morphology with uniform particle size, a hydrodynamic diameter of 76.2 nm and an encapsulation efficiency of 90%. The cumulative drug release rate reached approximately 93% within 144 h. Cell experiments confirmed that such PLGA nanoparticles significantly improved the bioavailability and antitumor activity of hesperidin and reduced the viability of colorectal cancer HCT116 cells, with the optimal effect observed at the concentration of 10 μg/mL [88]. Nevertheless, all relevant experiments were limited to cellular models and lacked in vivo animal verification. Further extensive animal studies are therefore warranted to clarify whether PLGA-encapsulated hesperidin could ameliorate its bioavailability, safety, tumor-targeting performance, and other critical properties in vivo.
Hesperidin was encapsulated into nanoemulsions (HP-NEM) by spontaneous emulsification to improve solubility and enhance the therapeutic effect on breast cancer. Physicochemical characterization showed that the optimized HP-NEM was spherical with a particle size of 305 ± 40.8 nm, a polydispersity index of 0.308 ± 0.04 and an encapsulation efficiency of 93 ± 0.45%. Within 48 h, the cumulative drug release of HP-NEM reached 98.57 ± 0.39% (w/w), whereas free hesperidin only exhibited a cumulative release of 46.35 ± 0.61% (w/w). In vitro experiments verified that HP-NEM exerted selective cytotoxicity on breast cancer MCF-7 cells and had no obvious toxicity to normal HEK293 cells [45]. Hesperidin nanoemulsions have also shown favorable anticancer effects in prostate cancer research [89]. Nevertheless, extensive in vivo studies are still warranted to systematically evaluate the bioavailability, biosafety and targeting capability of HP-NEM.
A comprehensive comparison of the three modification strategies is as follows: hesperidin methyl chalcone improves lipophilicity, but only its renoprotective effect has been verified and its antitumor activity remains unknown; PLGA nanoparticles have high encapsulation efficiency and excellent sustained release performance, but lack satisfactory active targeting ability; hesperidin nanoemulsions significantly enhance solubility and exert selective toxicity to tumor cells, while their in vivo stability needs to be optimized. Currently, all delivery systems are still in the stage of cellular and animal experiments and have not been applied in clinical research.

4.2. Spatiotemporal Specificity Regulation: Avoiding Damage to Normal Stem Cells

While killing tumor cells, the multi-target characteristics of hesperidin may interfere with the function of normal stem cells. pH-responsive nanocarriers can realize targeted drug release in the tumor microenvironment and reduce adverse effects on normal tissues and stem cells, which is a feasible direction to achieve spatiotemporal-specific regulation [90,91]. However, relevant research reports are limited, and the biosafety and in vivo targeting efficiency of carriers need verification. Therefore, the establishment of tissue-specific delivery methods or conditionally activated prodrugs can improve the accuracy of spatiotemporal specificity during hesperidin intervention [92].

4.3. Management of CSC Heterogeneity: Improving Therapeutic Consistency

The prominent heterogeneity of CSCs is a major obstacle for the application of hesperidin. Studies have shown that although hesperidin can inhibit the proliferation of prostate cancer cells, its pharmacological efficacy varies greatly among different tumor cell subpopulations [89]. At present, single-cell technology has been applied to clarify the heterogeneity of primary neuroblastoma, leukemia and CSCs in the tumor microenvironment [93,94]. In addition, tumor organoid technology is used to recapitulate the heterogeneity of hepatocellular carcinoma, gastric cancer and other malignant tumors [95,96]. Although these technologies have not been applied in research on hesperidin and hesperetin, they provide references for solving the problem of CSC heterogeneity in the future.

4.4. Safety Evaluation and Dose Optimization: Defining the Therapeutic Window

Preclinical studies have demonstrated that hesperidin exerts prominent antitumor effects at effective dosages. However, the long-term effects of long-term administration on normal stem cells and the synergistic toxicity of combined medication still need further evaluation [97,98]. The PharmaFormer artificial intelligence drug prediction model established by Zhou Yuru and colleagues integrates a large number of preclinical models via transfer learning and constructs an innovative framework for predicting clinical drug responses. Pharmacokinetic–pharmacodynamic models can be used to optimize the dosage [99]. Although this model has not been applied in the research of hesperidin and hesperetin, it provides a direction for establishing a dosage optimization model for hesperidin based on the PharmaFormer framework.

5. Conclusions and Future Perspectives

This review summarizes the antitumor mechanisms of hesperidin and its in vivo metabolite hesperetin. This provides a theoretical basis for the hypothesis that hesperidin and hesperetin may interfere with core signaling pathways of CSCs via epigenetic modifications, thereby suppressing the self-renewal, invasion and metastasis of cancer stem cells and reversing chemoresistance in tumor cells. Meanwhile, it offers a novel perspective for future investigations into precise molecular mechanisms underlying hesperidin and hesperetin in the regulation of tumor progression. Multiple in vitro and in vivo experiments have confirmed that the combination of these two compounds with conventional chemotherapeutic drugs exerts prominent synergistic antitumor effects with low intrinsic toxicity.
At present, many problems remain to be solved in this research field. Firstly, the molecular network of crosstalk between epigenetic regulation and CSCs modulated by hesperidin and hesperetin has not been fully elucidated, and potential new targets such as TOP2A, m6A and FOXP3 need further verification. Secondly, poor lipophilicity and low oral bioavailability restrict the in vivo efficacy of the compounds. Thirdly, CSC heterogeneity and the potential impacts of drugs on normal stem cells have not been fully evaluated.
Future research can be carried out from three aspects: (1) combining single-cell epigenomics and tumor organoid models to analyze the specific mechanisms of drug action in different tumors; (2) optimizing pharmaceutical technologies such as nanodelivery systems and responsive prodrugs to improve drug targeting ability and bioavailability; and (3) conducting long-term toxicity assessment and safety evaluation of combined medication, optimizing administration dosage combined with artificial intelligence models, and promoting the clinical transformation of these natural flavonoids.

Author Contributions

M.G.: Conceptualization, methodology, investigation, data curation, writing—original draft, and visualization. L.S. (Linxin Shao): Investigation, formal analysis, and writing—review and editing. H.Y.: Resources, visualization, and writing—review and editing. Q.K.: Methodology, validation, and formal analysis. L.S. (Lele Shang): Investigation, data curation, and visualization. H.G.: Resources, funding acquisition, and writing—review and editing. F.L.: Conceptualization, supervision, project administration, funding acquisition, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Programs from the National Natural Science Foundation of China (82260530), the Scientific Research Project of Shaanxi Provincial Department of Education (24JK0726) and Yan’an Key Research and Development Program Project (2025SLSFGG-124).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Acknowledgments

During the preparation of this manuscript, the authors used DeepL Translator (Version 2026) for English translation and linguistic polishing, EndNote (Version 2025) for reference management and citation formatting, and Adobe Illustrator (Version 2026) for the design and editing of schematic figures. All authors have reviewed and edited the manuscript content and assume full responsibility for this publication. Additionally, all figures in this work are original and have not been previously published by any party, so no copyright authorization is needed.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

ALDH1Aldehyde dehydrogenase 1
BCL-2B-cell lymphoma 2
CDCluster of differentiation
CDH1Cadherin 1 (E-cadherin)
CDH2Cadherin 2 (N-cadherin)
CK-19Cytokeratin 19
CSCsCancer stem cells
CXCR-4C-X-C chemokine receptor type 4
DNMTsDNA methyltransferases
DOT1LDOT1-like histone lysine methyltransferase
EACEhrlich ascites carcinoma
EMTEpithelial–mesenchymal transition
FN1Fibronectin 1
FOXP3Forkhead box P3
GSHGlutathione
HDACsHistone deacetylases
HIF-1αHypoxia-inducible factor-1α
HMCHesperidin methyl chalcone
HP-NEMHesperidin nanoemulsions
ICAM-1Intercellular adhesion molecule 1
IFN-γInterferon-gamma
IL-1βInterleukin-1 beta
IL-6Interleukin-6
IκBInhibitor of nuclear factor kappa-B
JAK-STATJanus kinase-signal transducer and activator of transcription
Ki-67Marker of proliferation Ki-67
lncRNALong non-coding RNA
LGR5Leucine-rich repeat-containing G-protein coupled receptor 5
MDAMalondialdehyde
MDM2Mouse double minute 2 homolog
miRNA/miRMicroRNA
MLH1MutL homolog 1
MMPMatrix metalloproteinase
MSH2MutS homolog 2
MYC/c-MycMYC proto-oncogene
NANOGNanog homeobox
NF-κBNuclear factor kappa-B
NotchNotch signaling pathway
Nrf2Nuclear factor erythroid 2-related factor 2
OCT4Octamer-binding transcription factor 4
OSCCOral squamous cell carcinoma
p21Cyclin-dependent kinase inhibitor 1A
p53Tumor protein p53
PI3K/AKTPhosphatidylinositol 3-kinase/Protein kinase B
PLGAPoly(lactic-co-glycolic acid)
PPARγPeroxisome proliferator-activated receptor gamma
PTXPaclitaxel
ROSReactive oxygen species
SDF-1αStromal cell-derived factor-1 alpha
SOX2SRY-box transcription factor 2
SurvivinBaculoviral IAP repeat-containing protein 5
TGF-β/SMADTransforming growth factor-beta/Smad signaling pathway
TNF-αTumor necrosis factor-alpha
TOP2ADNA topoisomerase II alpha
TRPM2Transient receptor potential melastatin 2
VEGFVascular endothelial growth factor
VimentinVimentin cytoskeletal protein
Wnt/β-cateninWnt/β-catenin signaling pathway
XIAPX-linked inhibitor of apoptosis protein
ZEB2Zinc finger E-box binding homeobox 2
ΔΨmMitochondrial membrane potential
m6AN6-methyladenosine
CpGCytosine-phosphate-Guanine
5-FU5-fluorouracil

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Figure 1. Direct and indirect epigenetic cascades mediating the antitumor activities of hesperetin and hesperidin across gastric and breast cancers and B lymphoblastic leukemia. Their tumor-suppressive functions rely on histone/non-coding RNA epigenetic regulation, as well as modulation of MLH1/MSH2 and PPARγ to induce cell cycle arrest and apoptosis. Green up-arrows: upregulated molecules; green down-arrows: downregulated molecules; red bars: molecular inhibition.
Figure 1. Direct and indirect epigenetic cascades mediating the antitumor activities of hesperetin and hesperidin across gastric and breast cancers and B lymphoblastic leukemia. Their tumor-suppressive functions rely on histone/non-coding RNA epigenetic regulation, as well as modulation of MLH1/MSH2 and PPARγ to induce cell cycle arrest and apoptosis. Green up-arrows: upregulated molecules; green down-arrows: downregulated molecules; red bars: molecular inhibition.
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Figure 2. Putative multi-pathway networks underlying the anti-stemness, anti-proliferative and anti-metastatic activities of hesperetin and hesperidin in breast, lung and liver cancer models. Hesperetin triggers G2/M arrest and lung cancer apoptosis via Notch1/ER stress. Hesperidin suppresses CSC phenotypes, cell cycle progression and EMT through Wnt/β-catenin, PI3K/AKT, CXCR4/NF-κB and c-Myc G-quadruplex signaling. Green up-arrows: upregulated molecules; green down-arrows: downregulated molecules; red bars: molecular inhibition.
Figure 2. Putative multi-pathway networks underlying the anti-stemness, anti-proliferative and anti-metastatic activities of hesperetin and hesperidin in breast, lung and liver cancer models. Hesperetin triggers G2/M arrest and lung cancer apoptosis via Notch1/ER stress. Hesperidin suppresses CSC phenotypes, cell cycle progression and EMT through Wnt/β-catenin, PI3K/AKT, CXCR4/NF-κB and c-Myc G-quadruplex signaling. Green up-arrows: upregulated molecules; green down-arrows: downregulated molecules; red bars: molecular inhibition.
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Figure 3. Schematic overview of confirmed and putative epigenetic-CSC regulatory networks modulated by hesperetin and hesperidin. Hesperidin directly suppresses Wnt/β-catenin and miR-132/ZEB2 cascades to induce tumor cell apoptosis and restrain CSC properties. Multiple unvalidated epigenetic axes centered on TOP2A and FOXP3 are hypothesized to bridge hesperetin/hesperidin activity with CSC homeostasis, as marked by red question marks. Green arrows denote altered molecular expression; red bars indicate molecular inhibition.
Figure 3. Schematic overview of confirmed and putative epigenetic-CSC regulatory networks modulated by hesperetin and hesperidin. Hesperidin directly suppresses Wnt/β-catenin and miR-132/ZEB2 cascades to induce tumor cell apoptosis and restrain CSC properties. Multiple unvalidated epigenetic axes centered on TOP2A and FOXP3 are hypothesized to bridge hesperetin/hesperidin activity with CSC homeostasis, as marked by red question marks. Green arrows denote altered molecular expression; red bars indicate molecular inhibition.
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Table 1. Synergistic antitumor effects and molecular mechanisms of hesperidin/hesperetin combined with chemotherapeutics in diverse tumors.
Table 1. Synergistic antitumor effects and molecular mechanisms of hesperidin/hesperetin combined with chemotherapeutics in diverse tumors.
Tumor TypeCombined Chemotherapeutic DrugCore Biological EffectsKey Molecular Regulatory Mechanisms
Oral squamous cell carcinoma (OSCC)Paclitaxel (PTX)Reduce tumor cell viability, increase apoptotic cell proportion, suppress cell migration; achieve high tumor inhibition rate superior to PTX monotherapyUpregulate Bax and Caspase-3 to trigger apoptosis; downregulate EMT-related MMP-9 and N-cadherin, upregulate E-cadherin to inhibit EMT and cell migration [33]
Esophageal carcinoma (Eca-109 cells)5-Fluorouracil (5-FU)Better anti-proliferative and anti-invasive effects than single drug; induce tumor cell apoptosis1. Arrest cells at G0/G1 phase, inhibit PI3K/AKT pathway, upregulate p21, downregulate Cyclin D1, MMP-2 and MMP-9; 2. activate mitochondrial apoptotic pathway, reduce Bcl-2/Bax ratio, activate Caspase-3/9 [34]
Malignant melanoma (A431 cells); Ehrlich ascites carcinoma (EAC) mouse modelCisplatinSynergistically reduce cell viability; enhance proliferation inhibition and apoptosis induction compared with monotherapy1. In EAC model: downregulate proliferation marker Ki-67, upregulate Caspase-3; 2. in A431 cells: upregulate pro-apoptotic Bax, Caspase-3/7, downregulate anti-apoptotic Survivin [35,36]
Laryngeal squamous cell carcinoma (Hep-2 cells)CisplatinStrengthen cisplatin-mediated cytotoxicity; aggravate oxidative damage and induce massive tumor cell apoptosisActivate TRPM2 channel to trigger extracellular Ca2+ overload and calcium homeostasis disorder; induce ROS accumulation, GSH depletion and MDA elevation to exacerbate oxidative stress and lipid peroxidation; decrease mitochondrial membrane potential to cause mitochondrial dysfunction; upregulate pro-inflammatory IL-1β and TNF-α [37]
Human endometrial adenocarcinoma (ISHIKAWA cells)GemcitabineSynergistically inhibit tumor proliferation; exert dual effects of chemosensitization and reduce chemotherapy-induced oxidative toxicity1. Modulate mitochondrial apoptosis: upregulate Bax, downregulate Bcl-2, activate Caspase-3/7; 2. downregulate HIF-1α and VEGF to block tumor angiogenesis and hypoxia adaptation; 3. scavenge excessive ROS to relieve oxidative injury induced by gemcitabine [38]
Cervical cancer (HeLa cells)DoxorubicinPromote apoptosis of cervical cancer cells; reduce inflammatory responseUpregulate Caspase-3; downregulate Bcl-2, pro-inflammatory cytokines IL-1β, IL-6 and TNF-α [39]
Hepatocellular carcinoma (HepG2 cells)SorafenibImprove chemosensitivity; inhibit cancer stem cell stemness; suppress cell proliferation and induce apoptosisDownregulate CSC markers CD44, CD133 and β-catenin; upregulate Caspase-3 [40]
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Guo, M.; Shao, L.; Yin, H.; Kou, Q.; Shang, L.; Guan, H.; Li, F. Hesperidin and Hesperetin: Epigenetic-Stemness Crosstalk, Antitumor Mechanisms, Preclinical Data and Translation Barriers. Biomolecules 2026, 16, 1063. https://doi.org/10.3390/biom16071063

AMA Style

Guo M, Shao L, Yin H, Kou Q, Shang L, Guan H, Li F. Hesperidin and Hesperetin: Epigenetic-Stemness Crosstalk, Antitumor Mechanisms, Preclinical Data and Translation Barriers. Biomolecules. 2026; 16(7):1063. https://doi.org/10.3390/biom16071063

Chicago/Turabian Style

Guo, Mengqi, Linxin Shao, Huiqing Yin, Qianrui Kou, Lele Shang, Haixia Guan, and Fang Li. 2026. "Hesperidin and Hesperetin: Epigenetic-Stemness Crosstalk, Antitumor Mechanisms, Preclinical Data and Translation Barriers" Biomolecules 16, no. 7: 1063. https://doi.org/10.3390/biom16071063

APA Style

Guo, M., Shao, L., Yin, H., Kou, Q., Shang, L., Guan, H., & Li, F. (2026). Hesperidin and Hesperetin: Epigenetic-Stemness Crosstalk, Antitumor Mechanisms, Preclinical Data and Translation Barriers. Biomolecules, 16(7), 1063. https://doi.org/10.3390/biom16071063

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