TO THE EDITOR
We read with great interest the recent article by Tong et al[1] published in the World Journal of Gastroenterology, detailing the synergistic anti-cancer effects of triptolide (TP) and quercetin in hepatocellular carcinoma (HCC). This study significantly contributes to the combination chemotherapy for HCC, a malignancy characterized by high morbidity and mortality rates globally[2,3]. Although the therapeutic landscape for advanced HCC has progressed substantially over the past decade, many patients exhibit primary or acquired resistance to existing treatments, highlighting the urgent need for novel therapeutic strategies[4,5]. In this context, Tong et al’s study[1], which investigates a combination of natural products, is both timely and highly relevant. We here offer comprehensive commentary on the significant merits of the study and highlight areas warranting further investigation to facilitate its potential clinical translation.
Merits of the study: A multifaceted and mechanistic approach
Tong et al’s study[1] has a rigorous and multidimensional research design. The authors skillfully integrated in vivo and in vitro models to build a compelling case for combining TP and quercetin. In vivo, the use of a HepG2 cell subcutaneous xenograft model provided strong evidence that the combination therapy significantly inhibited tumor growth. This was complemented by a comprehensive suite of in vitro assays (CCK-8, colony formation, wound healing, Transwell migration, invasion, and flow cytometry), which collectively demonstrated the potent inhibitory effects of the combination on HCC cells. This dual in vivo/in vitro validation strategy is necessary for substantiating therapeutic claims, particularly for a disease as heterogeneous as HCC[4].
Moreover, the study meticulously identified an optimal synergistic dose combination (25 nmol/L TP + 100 μmol/L quercetin) via cell viability assays and analysis using CompuSyn software. A resulting combination index < 1 provides quantitative evidence of a strong synergistic interaction. This is a crucial finding, as it addresses the major limitations of TP monotherapy: Its narrow therapeutic window and significant toxicity. By combining TP with quercetin, the authors actualized the principle of “synergistic efficacy and toxicity reduction”. This approach not only maximizes the anti-tumor effect but also facilitates the application of TP at a lower, less toxic dose. This strategy aligns with modern pharmacological goals, where combination therapies are designed to improve efficacy while mitigating adverse effects, a concept also investigated with other natural compounds such as Astragalus polysaccharides[6]. The added benefit of the hepatoprotective properties of quercetin further increases the clinical potential of this combination, making it particularly suitable for patients with HCC, who often have compromised liver function[7].
The most commendable aspect of the study is probably its comprehensive investigation of the underlying molecular mechanisms. Through transcriptomic analysis, the authors identified the Janus kinase/signal transducer and activator of transcription (JAK-STAT) and mammalian target of rapamycin (mTOR) signaling pathways as key targets of combination therapy. Both pathways are well-established drivers of hepatocarcinogenesis. The JAK-STAT pathway is frequently implicated in the proliferation and survival of HCC cells[2,8], and it is activated in specific genetic subtypes of HCC[9]. Similarly, the phosphoinositide 3-kinases (PI3K)/protein kinase B/mTOR pathway is a central regulator of cell growth and is often hyperactivated in HCC, contributing to the progression and metastasis of tumors[6,10,11]. In the study, these pathways were significantly modulated only by the combination therapy, and not by either drug alone, strongly suggesting a multitarget, multi-pathway synergistic mechanism. This finding is further supported by molecular docking analyses showing that both TP and quercetin can bind to key proteins within these pathways, such as JAK1, STAT3, PI3K, and mTOR (Figure 1). This multipronged attack on critical oncogenic signaling networks is a promising strategy to overcome the signaling redundancy and therapeutic resistance associated with single-agent treatments in HCC[12,13].
Figure 1 Model showing how triptolide and quercetin regulate the Janus kinase-signal transducer and activator of transcription and mammalian target of rapamycin signaling pathways.
This schematic illustrates the molecular mechanisms through which triptolide and quercetin may exert their anticancer effects. Both triptolide and quercetin can physically bind to Janus kinase 1, signal transducer and activator of transcription 3, phosphoinositide 3-kinases and mammalian target of rapamycin proteins. This multitarget inhibition disrupts oncogenic signaling. JAK1: Janus kinase; STAT3: Signal transducer and activator of transcription; PI3K: Phosphoinositide 3-kinases; AKT: Protein kinase B; mTOR: Mammalian target of rapamycin; PIP2: Phosphatidylinositol 4,5-bisphosphate; PIP3: Phosphatidylinositol-3,4,5-trisphosphate.
Limitations and future directions
Despite its considerable strengths, this study has several limitations that should be addressed in the future to enable clinical application. First, the reliance on an immunodeficient mouse model, while standard for xenograft studies, prevents researchers from evaluating the interaction of the therapy with the host immune system. The tumor microenvironment in HCC is a complex ecosystem of immune cells, stromal cells, and the extracellular matrix that strongly influences tumor progression and the response to therapy[4,5]. Natural products, including polysaccharides and flavonoids such as quercetin, exert immunomodulatory effects, such as reshaping the polarization of macrophages and regulating the functions of T cells[6]. Therefore, the full therapeutic potential of the combination of TP and quercetin, which may involve synergistic activation of anti-tumor immunity, could be underestimated in an immunodeficient setting. Further studies need to be conducted using syngeneic or humanized mouse models to assess these immunomodulatory dimensions and the efficacy of the combination in a more clinically relevant context[14].
Second, the poor pharmacokinetics and low bioavailability of many natural products are significant hurdles to their clinical translation[6]. The study does not provide data on pharmacokinetics or bioavailability, making it difficult to correlate the effective in vitro and in vivo doses with clinically achievable concentrations. A systematic investigation into the absorption, distribution, metabolism, and excretion properties of TP and quercetin, when administered in combination, is a critical next step. Furthermore, investigating advanced drug delivery systems, such as lipid nanoparticles or polymer nanoparticles, can help overcome these limitations by improving their solubility, stability, and targeted delivery to the tumor site[5,15].
Third, the combination of TP and quercetin can inhibit the JAK-STAT and mTOR signaling pathways, thereby exerting anti-tumor effects, which could be determined by transcriptomics and western blot assays. However, the causal relationship of this mechanism has not been further validated in animal models in vivo. Specifically, the authors did not demonstrate the anti-tumor effects of the two drugs using activators of the JAK-STAT or mTOR signaling pathways (such as interleukin-6, interleukin-22, or mTOR agonists)[16-19] to “reverse” or “compensate” for the effects, thus proving that the inhibition of these two signaling pathways is the key mechanism underlying the combination therapy. Transcriptome analysis revealed simultaneous regulation of the JAK-STAT and mTOR pathways in the combination therapy group, whereas these pathways remained inactive in the monotherapy group. This finding indicates that the two drugs may achieve their effects through synergistic actions on multiple targets and pathways. However, further validation is needed to elucidate the specific functions of these pathways in vivo. If activation of the JAK-STAT or mTOR pathway by activators in mouse models leads to a weakening or failure of the anti-tumor effects of combination therapy, it would provide stronger evidence of the key roles played by these signaling pathways in combination therapy.
Additionally, the study did not assess the time and dose dependence of the dynamic changes in the signaling pathway. Future studies should include pharmacodynamic assessments to correlate the kinetics of pathway inhibition with the durable anti-tumor response. Moreover, the connections among signaling pathway regulation, the tumor microenvironment, and the immune response have not been investigated. Therefore, these areas warrant further exploration.
Finally, as with all preclinical research, clinical trials must provide the ultimate validation. The journey from a promising preclinical combination to an approved therapy is long and requires rigorous evaluation of long-term safety, potential side effects, and efficacy in human patients. The current standard of care for advanced HCC includes tyrosine kinase inhibitors such as sorafenib and lenvatinib, as well as immune checkpoint inhibitors[20,21]. Future clinical development of the combination of TP and quercetin would need to define its place within this established therapeutic framework, perhaps as a treatment for patients resistant to standard therapeutic methods or in combination with existing agents.
Conclusion
Tong et al[1] provide a robust preclinical foundation for the combination use of TP and quercetin in HCC therapy. The authors have convincingly demonstrated synergistic anti-tumor activity through a well-designed series of experiments and provided insights into the plausible multi-pathway mechanism underlying this synergy. Both TP and quercetin can physically bind to key protein nodes within these cascades, including JAK1, STAT3, PI3K, and mTOR. We have summarized the multi-target inhibitory effects that disrupt carcinogenic signaling pathways in Figure 1. This study is a valuable addition to the growing body of research on natural product-based combination therapies for cancer[1].
To advance this promising start, we encourage researchers to focus on addressing the existing limitations. Investigations using immunocompetent animal models, detailed pharmacokinetic and bioavailability studies, and deeper mechanistic validation are essential next steps. Such efforts may facilitate the translation of this promising laboratory finding into a viable clinical strategy that can one day benefit patients with HCC, a goal that aligns with the broader push toward precision and personalized medicine in oncology[4]. We commend the authors for their excellent work and expect further development in this exciting area of research.
