Unlocking the potential of Calculus bovis: A breakthrough in liver cancer treatment via Wnt/β-catenin pathway modulation

Abstract

Liver cancer remains a significant global health challenge, characterized by high incidence and mortality rates. Despite advancements in medical treatments, the prognosis for liver cancer patients remains poor, highlighting the urgent need for novel therapeutic approaches. Traditional Chinese medicine (TCM), particularly Calculus bovis (CB), has shown promise in addressing this need due to its multi-target therapeutic mechanisms. CB refers to natural or synthetic gallstones, traditionally sourced from cattle, and used in TCM for their anti-inflammatory, detoxifying, and therapeutic properties. In modern practice, synthetic CB is often utilized to ensure consistent supply and safety. This article aims to discuss the findings of Huang et al, who investigated the anti-liver cancer properties of CB, focusing on its ability to inhibit M2 tumor-associated macrophage (TAM) polarization via modulation of the Wnt/β-catenin pathway. Huang et al employed a comprehensive approach integrating chemical analysis, animal model testing, and advanced bioinformatics. They identified active components of CB using UPLC-Q-TOF-MS, evaluated its anti-neoplastic effects in a nude mouse model, and elucidated the underlying mechanisms through network pharmacology, transcriptomics, and molecular docking studies. The study demonstrated that CB significantly inhibited liver tumor growth in vivo, as evidenced by reduced tumor size and weight in treated mice. Histological analyses confirmed signs of tumor regression. CB was found to modulate the tumor microenvironment by inhibiting the polarization of M2 phenotype-TAMs, as shown by reduced expression of M2 markers and downregulation of mRNA levels of C-C motif chemokine 22, arginase-1, transforming growth factor-beta 2, and interleukin-10. The study further revealed that CB’s antineoplastic activity involved the downregulation of Wnt5B and β-catenin and upregulation of Axin2, thus inhibiting the Wnt/β-catenin signaling pathway. These findings highlight the therapeutic potential of CB in liver cancer treatment through its modulation of the Wnt/β-catenin pathway and suppression of M2 phenotype-TAM polarization. This study underscores the value of integrating TCM with modern therapeutic strategies to develop novel effective treatments for liver cancer.

Key Words: Calculus bovis; Liver cancer; M2 phenotype tumor-associated macrophages; Wnt/β-catenin pathway; Traditional Chinese medicine; Tumor microenvironment; Antineoplastic therapy

Core Tip: This article discusses the significant findings of Huang et al, who demonstrated that Calculus bovis, a traditional Chinese medicine, exhibits potent anti-liver cancer effects. The study revealed that Calculus bovis effectively inhibits M2 tumor-associated macrophage polarization by modulating the Wnt/β-catenin pathway, leading to reduced tumor growth and improved tumor regression in vivo. These insights underscore the potential of integrating traditional Chinese medicinal approaches with modern therapeutic strategies to develop novel, multi-targeted treatments for liver cancer, addressing a critical need in oncology.

TO THE EDITOR

Liver cancer is a formidable global health challenge, ranking sixth in incidence and fourth in cancer-related mortality worldwide[1]. Despite advancements in medical science, the aggressive nature of liver cancer, characterized by rapid progression and poor prognosis, underscores the urgent need for innovative and effective treatment strategies[2]. Traditional treatment options, including surgical interventions such as liver resection or transplantation, interventional therapies, local ablation treatments, and targeted immunotherapies, have shown limited efficacy, especially in advanced stages of the disease[3-6]. The five-year survival rate for liver cancer patients remains dismally low at approximately 3%, highlighting the necessity for novel therapeutic approaches[7-10]. Traditional Chinese medicine (TCM) offers a promising alternative, characterized by its multi-component and multi-target therapeutic mechanisms[11,12]. TCM has been increasingly recognized for its potential in cancer treatment, providing a holistic approach that not only targets the tumor but also modulates the tumor microenvironment (TME)[13]. Within this context, Calculus bovis (CB), a valuable agent in TCM, has gained attention for its anti-tumor properties[14]. CB refers to natural or synthetic gallstones, traditionally sourced from cattle, and used for their purported anti-inflammatory, detoxifying, and therapeutic effects in TCM. In modern practice, synthetic versions of CB are often employed to ensure consistent supply and safety, and CB has been a critical component in compound formulations like Gou et al[15], historically utilized for liver cancer treatment[16].

However, its specific impact on the liver cancer microenvironment, particularly on tumor-associated macrophages (TAMs), remains underexplored[17]. TAMs, particularly the M2 phenotype-TAMs (M2-TAMs), play a crucial role in tumor progression by promoting tumor growth, metastasis, and angiogenesis[18]. These macrophages are known to facilitate an immunosuppressive environment through pathways such as nuclear factor kappa B, interleukin-6/signal transducers and activators of transcription 3, and Wnt/β-catenin[19-21]. Reversing TAM polarization from the tumor-promoting M2-TAMs to the tumor-inhibiting M1 phenotype represents a promising therapeutic strategy for cancer treatment[22]. In the article written by Huang et al[23], published in recently, the authors explore the anti-liver cancer effects of CB with a focus on its ability to modulate the Wnt/β-catenin pathway and inhibit M2-TAM polarization. This article aims to highlight the significance of these findings, discussing the potential mechanisms through which CB exerts its effects and the implications for future liver cancer therapies.

The comprehensive approach adopted in this study, encompassing the identification of active CB components using advanced mass spectrometry, evaluation of anti-neoplastic effects in animal models, and elucidation of underlying mechanisms via network pharmacology, transcriptomics, and molecular docking, provides a robust framework for understanding the therapeutic potential of CB[24,25]. By shedding light on the interaction between CB and the Wnt/β-catenin pathway, this study offers valuable insights into the development of novel anti-cancer strategies that leverage TCM.

THERAPEUTIC POTENTIAL OF CB IN LIVER CANCER

The study by Huang et al[23] provides compelling evidence supporting the anti-liver cancer properties of CB. Their findings demonstrate a multifaceted approach that integrates chemical analysis, animal model testing, and advanced bioinformatics to unravel the mechanisms underlying CB’s therapeutic effects[23].

INHIBITION OF LIVER TUMOR GROWTH

One of the primary findings of this study is the ability of CB to significantly inhibit liver tumor growth in vivo. The authors used a nude mouse model and demonstrated that treatment with CB led to a substantial reduction in tumor size and weight compared to the control group[23]. This antineoplastic effect was corroborated by histological analyses showing signs of tumor regression, such as reduced nucleolar staining intensity and the presence of nuclear fragments, particularly at medium and high doses of CB[23]. These results highlight the potent anticancer effects of CB in a preclinical setting, underscoring its potential as a therapeutic agent for liver cancer[23].

MODULATION OF TAMS

A pivotal aspect of the study is the investigation into how CB influences the TME, specifically TAMs. TAMs are known to exist in different phenotypic states, with M2-TAMs promoting tumor growth and metastasis through immunosuppressive pathways[23]. Huang et al[23] showed that CB effectively inhibits the polarization of M2-TAMs both in vitro and in vivo. This was demonstrated through flow cytometry analysis which revealed a reduction in M2-TAM markers such as CD206 and significant downregulation of mRNA levels of C-C motif chemokine 22, arginase-1, transforming growth factor-beta 2, and interleukin-10[23]. These findings are crucial as they suggest that CB can shift the balance towards a tumor-inhibiting microenvironment by suppressing M2-TAM polarization[23].

WNT/Β-CATENIN PATHWAY MODULATION

The study provides a detailed exploration of the molecular mechanisms behind CB’s effects, particularly focusing on the Wnt/β-catenin pathway. This pathway is known for its role in cell proliferation, differentiation, and apoptosis, and its dysregulation is often associated with cancer progression[23]. Huang et al[23] employed network pharmacology, transcriptomics, and molecular docking to demonstrate that CB’s antineoplastic activity involves modulation of the Wnt signaling pathway. Specifically, CB was shown to downregulate the expression of Wnt5B and β-catenin while upregulating Axin2, thus inhibiting the Wnt/β-catenin signaling pathway[23]. These molecular changes were further confirmed in vivo, reinforcing the pathway-specific action of CB in suppressing liver cancer[23].

INTEGRATED APPROACH AND FUTURE IMPLICATIONS

The integrated approach used in this study, which combines UPLC-Q-TOF-MS for chemical analysis, animal models for functional validation, and advanced bioinformatics for elucidating mechanisms, provides a comprehensive framework for understanding the therapeutic potential of CB[23]. Identifying 22 active components in CB, including 11 detected in the bloodstream, highlights the complexity and potential efficacy of this TCM[23]. The findings suggest that CB’s capacity to modulate the TME and specific signaling pathways could be leveraged to develop novel, multi-targeted cancer therapies[23]. The study by Huang et al[23] lays the foundation for several promising research directions. A key area yet to be explored is whether CB could impact other cell types involved in liver cancer progression. In-depth investigations could uncover CB’s effects on tumor fibroblasts or endothelial cells, essential components of the TME. Identifying CB’s direct target on TAMs is also a crucial objective, and techniques like cellular thermal shift assay or drug affinity responsive target stability could help determine specific molecular interactions between CB and intracellular proteins.

Additionally, considering the relationship between Wnt signaling and epigenetic regulation, it would be valuable to investigate whether CB influences epigenetic modifications in TAMs. This could involve methylation assays or histone modification studies to fully elucidate CB’s impact on gene expression within the TME. Future research employing advanced single-cell technologies, such as single-cell RNA sequencing and spatial transcriptomics, may offer detailed insights into CB’s effects on individual TAM populations, revealing changes in their functional phenotypes. Ultimately, these insights could expand the clinical applications of CB, including its potential for combination therapies and its use in treating other cancers characterized by M2-TAM infiltration.

IMPLICATIONS FOR FUTURE RESEARCH

The study by Huang et al[23] opens several avenues for future research: (1) Component-specific studies: Further investigations are needed to identify the specific components of CB responsible for its anti-liver cancer effects and their individual contributions to the observed outcomes; (2) Clinical trials: While the preclinical results are promising, clinical trials are necessary to validate the efficacy and safety of CB in liver cancer patients; (3) Combination therapies: Exploring the potential synergistic effects of CB with existing anti-cancer therapies could enhance its therapeutic efficacy and overcome resistance mechanisms; and (4) Broader applications: Given the multi-targeted nature of CB, its potential applications in other cancer types and diseases involving macrophage polarization should be explored.

CONCLUSION

The results presented by Huang et al[23] are significant as they offer a promising perspective on the use of TCM, particularly CB, in the treatment of liver cancer. By elucidating the mechanisms through which CB inhibits M2-TAM polarization and modulates the Wnt/β-catenin pathway, this study lays the groundwork for future research aimed at integrating traditional and modern medicine to combat cancer. The study not only enhances our understanding of CB’s therapeutic effects but also opens new avenues for developing effective liver cancer treatments based on traditional medicinal practices.