Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal malignancies globally. Its high incidence and mortality rates are especially high in regions with endemic hepatitis B virus infection and a high prevalence of cirrhosis, posing a substantial burden to global public health [1, 2, 3].

Lenvatinib, a multi-targeted tyrosine kinase inhibitor, exerts anti-tumor effects by blocking kinases including vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR), thereby disrupting tumor angiogenesis and signaling pathways regulating cell proliferation. It is established as a first-line standard treatment for advanced HCC [4, 5, 6]. However, its effectiveness is limited by the development of resistance. The mechanism behind lenvatinib resistance is complex and multifactorial. Tumor cells can bypass the inhibitory effects of drugs by activating alternative signaling pathways, including c-MET and EGFR. As a result, tumor cells undergo epithelial-mesenchymal transition or increase their stemness, thereby enhancing their invasiveness and survival capabilities. Immunosuppressive cells and fibroblasts in the tumor microenvironment also contribute to the development of drug resistance in tumor cells [7, 8]. Consequently, research is focusing on developing strategies to enhance lenvatinib sensitivity and effective combination therapies for HCC treatment.

Quercetin, a naturally occurring flavonoid found in various vegetables, fruits, and medicinal herbs, exhibits diverse pharmacological properties such as anti-inflammatory, antioxidant, and anti-tumor effects [9, 10, 11]. Recent studies indicate that quercetin inhibits tumor growth through mechanisms including G2/M phase cell cycle arrest, induction of mitochondrial apoptosis, and suppression of epithelial-mesenchymal transition (EMT), invasion, and metastasis [12, 13, 14]. Furthermore, it modulates key oncogenic signaling pathways like PI3K/AKT, NF-$\kappa$B, and MAPK/ERK, demonstrating potential to sensitize cancer cells to conventional chemotherapy and targeted agents [15, 16, 17]. The study of quercetin combined with sorafenib in HCC showed that quercetin combined with sorafenib synergistically downregulates oncogenes such as TNF-$\alpha$, VEGF, and NF-$\kappa$B, significantly inhibits tumor growth, and induces cell apoptosis [18, 19]. Quercetin has also been found to increase drug sensitivity by promoting copper-dependent cell death in lenvatinib-resistant HCC cells [20]. However, further research is necessary to identify the specific targets and mechanisms underlying quercetin-mediated lenvatinib sensitization.

Network pharmacology analysis suggests that quercetin, as a multi-target natural product, influences numerous key factors in HCC pathogenesis, including EGFR, PIK3CA, matrix metalloproteinase-9 (MMP-9), signal transducer and activator of transcription 3 (STAT3), and B-cell lymphoma 2 (Bcl-2), impacting critical biological processes including cell proliferation, apoptosis, angiogenesis, and the tumor microenvironment [21, 22, 23]. A comprehensive exploration of the interactive effects of quercetin and lenvatinib within regulatory networks could illuminate the mechanistic basis for their potential combined antitumor activity.

This study utilized the human HCC cell line Huh7 and a nude mouse xenograft model to investigate the synergistic anti-tumor effects of quercetin combined with lenvatinib. We aimed to elucidate the potential mechanisms involving cell proliferation, apoptosis, invasion, metastasis, and associated molecular pathways, thereby providing novel experimental evidence and strategic options for clinical HCC combination therapy.

This research systematically elucidates the potential of quercetin, a dietary flavonoid, to significantly enhance the anti-tumor activity of lenvatinib in HCC. Our experimental models demonstrate that the combination therapy exhibits a synergistic suppression of key oncogenic traits — proliferation, clonogenicity, invasion, and migration — while simultaneously activating apoptotic pathways. Consistent calculation of Combination Index (CDI) values below one in both cellular and animal studies provided quantitative validation of this pharmacological synergy. Moving beyond phenotypic observation, our integrated strategy, which combines computational network pharmacology with molecular validation, pinpointed PTK2 (Focal Adhesion Kinase) as a critical shared target. This mechanistic insight not only offers a potential clinical strategy to address lenvatinib resistance but also delineates a specific molecular pathway underlying the combined effect.

As a multi-targeted tyrosine kinase inhibitor, Lenvatinib exerts anti-tumor effects by disrupting angiogenic signaling through VEGFR, FGFR, and PDGFR, as well as directly suppressing tumor cell proliferation via RET and KIT inhibition [4, 5, 6, 26]. However, its efficacy in advanced HCC is frequently limited by the development of resistance, mainly due to the pronounced heterogeneity of HCC and its complex stromal microenvironment [27]. While our data confirm the standalone anti-HCC properties of quercetin, its main value appears to lie in its role as a potent sensitizer. The compound significantly lowers the threshold for lenvatinib’s efficacy, transforming a modest cytostatic response into a powerful cytotoxic and anti-metastatic outcome.

The synergy we observed extends beyond a mere additive impact on cell numbers. It represents a coordinated attack on multiple core cancer capabilities. The combination regimen not only more effectively suppressed immediate proliferation but also critically undermined the tumor’s long-term replicative potential, as demonstrated by the drastic reduction in colony-forming ability. Equally important, invasive and migratory behaviors — key drivers of metastasis — were significantly impaired, alongside a marked shift in the cellular population towards apoptosis. The consistent CDI values obtained from independent in vitro and in vivo systems provide robust, multi-level confirmation of a precise synergistic interaction. This pharmacodynamic profile suggests a compelling clinical possibility that quercetin co-therapy could enhance lenvatinib’s therapeutic window, potentially maintaining tumor control at lower, less toxic drug doses [28]. Managing lenvatinib therapy is often complicated by common adverse effects, such as hypertension, proteinuria, and hand-foot syndrome, which can necessitate dose modifications and disrupt treatment continuity, thereby jeopardizing long-term outcomes [29, 30]. In this context, quercetin stands out as a uniquely suitable combination partner. Its status as a ubiquitous dietary component, along with a long history of safe use in humans, positions it as an ideal candidate for mitigating regimen-related toxicity. By synergistically enhancing anti-tumor potency, this combination could theoretically enable effective disease management with reduced lenvatinib exposure, ultimately improving patient tolerability and quality of life, a critical goal in palliative oncology.

The major finding of our work is the identification of PTK2 as a central mediator of this synergy. PTK2 acts as a non-receptor tyrosine kinase and serves as an important signaling hub that integrates inputs affecting cell survival, proliferation, and motility [31]. Its pathogenic role in HCC is well-documented, with numerous reports linking its overexpression to advanced disease stages, vascular invasion, and poor patient survival [32, 33]. Mechanistically, PTK2 sustains tumor cell viability by activating pro-survival cascades like PI3K/Akt/mTOR and Ras/MAPK [34, 35]. Concurrently, it functions as a master regulator of the invasive program, driving metastasis and shaping a permissive tumor microenvironment through effects on angiogenesis and immune evasion [36, 37]. Our results indicate that while both monotherapies can modestly attenuate PTK2 levels, their combination induces a far more substantial downregulation. This cooperative suppression likely disrupts a network of downstream oncogenic signals, ultimately manifesting as the observed potentiation of apoptosis (via Bcl-2/Bax) and reversal of EMT (via E-cadherin/N-cadherin). The congruent modulation of these pivotal pathways strongly supports PTK2’s role as an orchestrator of the synergistic response.

However, this study has some limitations, including the reliance on a single HCC cell line (Huh7) and a subcutaneous xenograft model. In the future study, more HCC cell lines will be applied to study the sensitization effect of quercetin on lenvatinib. Although PTK2 is identified as a key shared target, the precise upstream and downstream molecular pathways through which the combination therapy modulates PTK2 and exerts its synergistic effects require further elucidation. Future work must clarify the precise molecular events by which each agent, and their combination, regulates PTK2. Is control exerted at the transcriptional level, through mRNA stability, or via post-translational modification and protein degradation? From a translational perspective, it is essential to determine if tumors exhibiting high PTK2 expression or activation constitute a biomarker-defined population that derives exceptional benefit from this regimen, paving the way for personalized therapy. Furthermore, evaluating the efficacy of this combination in models with acquired lenvatinib resistance would offer invaluable insights for managing refractory disease.

In conclusion, this study establishes quercetin as a potent enhancer of lenvatinib’s anti-HCC efficacy. The observed synergy, which influences a range of cancer hallmarks from proliferation and survival to invasion and metastasis, is mechanistically rooted in the cooperative targeting of PTK2. These findings provide a strong scientific foundation for clinical exploration of the quercetin-lenvatinib combination, proposing a dual-pronged strategy to enhance therapeutic efficacy and potentially overcome resistance in HCC.

The data sets used or analyzed during the current study are available from the corresponding author upon reasonable request.

YZ designed the research study. JL and QHY performed the research. KL and XC collected and analyzed the data. JL and YZ have been involved in drafting the manuscript and all authors have been involved in revising it critically for important intellectual content. All authors give final approval of the version to be published. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.

The patient samples and information collected in the study were approved by the ethics committee of Hunan Provincial People’s Hospital/The First Affiliated Hospital of Hunan Normal University ([2024] -032). The study was carried out in accordance with the guidelines of the Declaration of Helsinki. All pathological samples obtained were informed to patients or their families/legal guardians and signed an informed consent form. The animal experiments in the study were approved by the ethics committee Hunan Provincial People’s Hospital/The First Affiliated Hospital of Hunan Normal University ([2023] -156). The animal experiments in the study was in compliance with the revised Animals (Scientific Procedures) Act 1986 in the UK and Directive 2010/63/EU in Europe).

Not applicable.

This work was financially supported by the following grant: Research Project of Hunan Provincial Health Commission (20254505).

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/FBL47715.