Harnessing the power of Calculus bovis: Anti-cancer properties and Wnt pathway modulation in hepatocellular carcinoma

Abstract

In this manuscript, we comment on the article, which explores the anti-cancer effects of Calculus bovis (CB) in tumor biology. We highlight its potential, particularly in hepatocellular carcinoma (HCC), where it inhibits the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways and induces apoptosis. CB contains compounds such as oleanolic acid and ursolic acid that target interleukin-6, mitogen-activated protein kinase 8, vascular endothelial growth factor, and caspase-3, offering anti-inflammatory and hepatoprotective benefits. The manuscript also discusses CB sativus (CBS), an artificial substitute, which has shown efficacy in reducing hepatic inflammation and oxidative stress in animal models. We emphasize the need for further research on the effects of CBS on the gut-liver axis and gut microbiota, and on targeting Wnt signaling and M2 tumor-associated macrophage as potential therapeutic strategies against HCC.

Key Words: Calculus bovis; Liver cancer; Tumor-associated macrophages; M2 polarization; Wnt signaling pathway

Core Tip: The traditional Chinese medicine Calculus bovis (CB) is known to affect tumor biology by showing promise in inhibiting hepatocellular carcinoma (HCC) progression by targeting the Wnt signaling pathway. The study highlights CB's anti-cancer properties, particularly against liver cancer, through modulation of pathways such as phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin, and compounds such as oleanolic acid and ursolic acid. Additionally, CB sativus has shown efficacy in reducing hepatic inflammation and oxidative stress, suggesting potential therapeutic strategies for HCC, warranting further clinical validation.

TO THE EDITOR

Traditional Chinese medicine (TCM) which originated 2000 years ago is represented by various practices that include herbal medicine, acupuncture, and dietary therapy. Herbal medicine is basically the main therapy in TCM and it involves the employment of natural plant, animal, and mineral substances to prevent and treat diseases. These medicines are often used in combination to enhance therapeutic effects and minimize side effects. From a western medical point of view, TCM compounds have an inhibitory effect on cancer cell growth and metastasis as well as promoting the immune system. Consequently, TCM compounds have the potential to become an essential part of cancer management[1]. The pathways through which CTM herbs act to produce their effects are diverse and complicated. These compounds can be involved in the modulation of various biological pathways, including the immune system, inflammatory processes, and cellular metabolism. The multifaceted nature of these herbs makes them effective in treating various ailments, including cancer[2,3]. Several Chinese herbs have gained attention due to their potential anticancer properties. Calculus bovis (CB), also known as natural bezoar, is a TCM derived from the gallbladders of cattle. This substance has traditionally been used for its anti-inflammatory, antipyretic, and anti-toxic effects. In recent years, scientific research has begun to uncover its potential as an anti-cancer agent, particularly in the context of liver cancer[4]. CB can suppress the growth of castration-resistant human pancreatic tumors by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin signaling pathways and inducing cell apoptosis[5]. Table 1 summarizes various Chinese herbal medicines and their active compounds that have been studied for their anticancer properties[6-24]. These herbs are used either alone or in combination with conventional therapies to enhance efficacy and reduce adverse effects. For example, Akula et al[25] showed that the anticancer effects of metformin were enhanced when combined with berberine and resulted in inhibition of the proliferation of pancreatic cancer cells.

Table 1 Chinese herbal medicines with anticancer properties.

Herb name	Active compound	Biological action	Cancer types treated	Ref.
Licorice root (glycyrrhizia uralensis)	Glycyrrhizin	Antioxidant activity, interferon production, T-cell activation in the liver, inhibition of Notch signaling pathway	HCC, triple negative breast cancer	[6,7]
Tripterygium wilfordii	Triptolide and celastrol	Tumour necrosis factor-α-induced apoptosis, suppression of NF-B activation, farnesoid X receptor antagonist, increase in autophagic flux	HCC, non-small-cell lung cancer	[8-10]
Curcuma longa L	Curcumin	Inhibition of extracellular matrix secretion, modulation of MMPs expression, inhibition of M2 polarization of tumor macrophages	Liver cancer, breast cancer, prostate cancer	[11-14]
Stephania tetrandra S	Tetrandrine	MMP2 and MMP9 downregulation, apoptosis induction	Cervical cancer, triple-negative breast cancer, lung cancer	[15-17]
Coptis chinensis	Berberine	Inhibition of activator protein-1 and cyclooxygenase-2, inhibition of interleukin-6/Janus Kinase/Signal transducer and activator of transcription 3 pathway, inhibition of Hedgehog signaling pathway	Colon cancer, gastric cancer	[18-20]
Sophora alopecuraides L	Oxymatrine	Inhibition of mitophagy-activated NOD-like receptor thermal protein domain associated protein 3 inflammasome, modulation of tumor microenvironment, Inhibition of myelocytomatosis proto-oncogene/programmed cell death ligand 1 pathway	Colorectal cancer, melanoma	[21,22]
Radix salviae miltiorrhizae	Salvianolic acid	Inhibition of phosphatidylinositol 3-kinase/AKT signaling pathway, modulation of epithelial-mesenchymal-transition via AKT/mammalian target of rapamycin pathway	Gastric cancer, lung cancer	[23,24]

MMP: Matrix metalloproteinase; AKT: Protein kinase B; HCC: Hepatocellular carcinoma.

CB AND ITS ANTICANCER EFFECTS IN LIVER CANCER

Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer accounting for 90% of cases. A number of etiologies are associated with HCC which include hepatitis B virus (HBV) and hepatitis C virus (HCV), alcohol, diabetes, obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). The role of CB in the treatment of liver cancer has garnered increasing interest due to its potential therapeutic properties. It has been known for a long time that CB has antipyretic, sedative, and anti-inflammatory responses[26]. Eleven compounds within CB were identified to demonstrate anti-liver cancer activity, among which oleanolic acid, ergosterol, and ursolic acid were highlighted. Notable potential targets for these compounds include interleukin (IL)-6, mitogen-activated protein kinase 8, vascular endothelial growth factor, and caspase-3. Previous findings revealed that the mechanism of action of CB involves immune-related and apoptosis-related pathways; however, the exact mechanism remains unexplored[27]. In a rat model of 17 α-ethynyl estradiol (EE) activated intrahepatic cholestasis, treatment with CB sativus (CBS), an artificial bioengineered substitute for CB significantly increased cumulative biliary excretions and elevated PDZ domain-containing 1 (PDZK1) mRNA and protein expressions. PDZK1 activates multi-drug resistance-associated protein 2 (MRP2) which improves biliary excretion. Treatment with CBS notably reversed the effects of EE by restoring the MRP2 and MRP4 expression levels which are the efflux pumps for the bile acid flow[28]. Wu et al[29], Liu et al[30] and Xiang et al[31] also suggested that this effect may be mediated through inhibition of the PI3K/AKT signaling pathway. In a recent study, it was depicted that CBS mitigated hepatic inflammation by suppressing the NF-B signaling pathway and reducing the tumour necrosis factor (TNF)-α, IL-1β, and IL-6 levels. Additionally, CBS alleviated oxidative stress through activation of the nuclear factor-E2-related factor 2 (Nrf2)-glutamate-cysteine ligase regulatory subunit/glutamate-cysteine ligase catalytic subunit gene and Nrf2-heme oxygenase-1 signaling pathways[32,33]. In addition to this, CBS also reduces basilar arterial production by repressing the enzymes viz, cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase and increasing CYP2B10, cytochrome P450 3A2, and human sulfotransferase 2A1 expression. CBS increases the nuclear translocation and protein expression of intestinal and hepatic farnesoid X receptor to control the expression of these enzymes and transporters[33]. Given the role of the gut-liver axis in cholestasis, future research is required for the investigation of how CBS affects gut microbiota composition which can subsequently impact liver health. He et al[34] utilized a fructose-induced steatosis model in LO2 hepatocytes to evaluate the lipid-lowering effects of CBS-containing serum through the Nrf2 pathway. CBS effectively reduced lipid droplet formation, intracellular triglyceride and reactive oxygen species levels, and hepatocyte apoptosis in rats. Also, the mRNA levels of sterol regulatory element binding protein-1c, carbohydrate responsive element binding protein, acetyl-coA carboxylase 1, caspase-3 and Bax were notably reduced with CBS treatment[34]. In addition to this, CBS is significantly used in the treatment of chronic liver disorders as in the study by He et al[35], which showed beneficial outcomes in a MASLD animal model by improving glucose and lipid metabolic conditions and repressing adipogenic protein expression. A comprehensive dose-response analysis would provide insights into the optimal therapeutic dosage for improving liver-associated metabolic disorders. Administration of cholic acid, a primary active component of CB has been shown to increase the serum levels of superoxide dismutase and decrease the levels of aspartate transaminase (AST), malondialdehyde (MDA), alanine transaminase (ALT), IL-6, and TNF-α thereby mitigating CCl4-induced liver damage, supporting the potential development of CA-based therapies for liver injury[36]. In a rodent model treated with alpha-naphthylisothiocyanate (ANIT) to induce cholestasis, treatment with CBS significantly reduced the effect of ANIT like altered bile flow and serum markers viz; alkaline phosphatase, AST, and ALT. Moreover, CBS effectively decreased the raised liver MDA levels[37]. Utilizing high-resolution mass spectrometry, transgenic zebrafish-based phenotypic screening, and mouse macrophage models, taurocholic acid (TCA) and glycocholic acid (GCA) were identified as potent and safe anti-inflammatory compounds derived from CB. Both bile acids demonstrated a significant reduction in lipopolysaccharide-induced macrophage recruitment and the secretion of proinflammatory cytokines/chemokines in both in vivo and in vitro models[38]. Treatment with CB effectively reduced γ-glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and alkaline phosphatase activities depicting its hepatoprotective property in the diethylnitrosamine murine model.

LIVER CANCER AND THE WNT SIGNALING PATHWAY- ROLE OF M2 TAMS

Inflammation is one of the frequent hallmarks of the tumor microenvironment (TME), and tumor macrophages (TAMs) are vital factors in the TME which are involved in inflammation-associated progression of HCC[39,40]. The risk factors including HBV, HCV, diabetes, and obesity trigger chronic liver injury leading to inflammation of hepatocytes. The crosstalk between macrophages and tumor cells is regulated through cell-cell interaction via various soluble factors, such as Wnt[41]. A murine based study showed that tumor-derived Wnt ligands induce M2 polarization by activating canonical Wnt/β-catenin signaling in macrophages, leading to tumor growth, metastasis, migration, and immunosuppression in HCC[42]. The M2 cells are known to secrete chemokines, while the HCC cells exhibit C-C chemokine receptors (CCRs), like CCR5 and CCR4[43], and are involved in modulating oncogenesis. A study by Zhu et al[44], explored the role of chemokine CCL17 in the tumorigenesis of HCC. The study revealed that higher levels of CCL17 are significantly related to pathological features of HCC and overall poor survival in patients. The ligand enhanced epithelial-mesenchymal-transition (EMT), Wnt/β-catenin signaling, and stemness in a liver cancer cell line[44]. Wnt signaling has been known to induce glycolysis in tumor cells and plays a vital role in modelling the immunocompromised microenvironment through cell-cell communication. Some factors like IL-10, transforming growth factor β (TGF-β), etc. in the TME stimulate Wnt2β expression in macrophages, which can assist the activation of glycolysis in HCC-TAMs through c-Myc, subsequently causing M2 polarization and promotes EMT, migration, and proliferation of malignant cells[45]. In addition to this, long non-coding RNAs (lncRNAs) that are reported to be associated with HCC are also found to be related to M2 polarization. A study explored the role of the lncRNA LINC00662 in HCC, and found that the lncRNA promotes HCC metastasis in both an autocrine and paracrine manner. LINC00662 regulates tumor cell invasion and cell cycle and represses apoptosis via Wnt/β-catenin signaling in an autocrine manner by competitively interacting with microRNA-107 (miR-107), miR-16, and miR-15a. On the other hand, in a paracrine manner, it activates M2 polarization in macrophages through Wnt/β-catenin signaling, further promoting tumor metastasis[46]. Another lncRNA, transcribed ultra-conserved RNA uc.306 is significantly upregulated in macrophages and is involved in the Hippo, Hedgehog, and Wnt signaling pathway, playing a significant role in HCC progression[47]. Moreover, other lncRNAs such as the focally amplified lncRNA on chromosome 1 and HOMER3-AS1, have been reported to modulate M2 polarization and activate Wnt/β-catenin signaling in HCC cells, hence enhancing HCC progression[48,49]. Altogether, this suggests that Wnt signaling plays an important role in M2 polarization of macrophages which are part of the TME and further interact with the tumor cells leading to tumor progression which is summarized in Figure 1. Targeting the pathways regulating M2 polarization or preventing TAMs polarization to the M2 stage could be crucial in impeding HCC progression.

Figure 1 The Wnt signaling pathway. A: Role of M2 macrophages in tumor progression: (1) Tumor cells induce the release of chemokines or cytokines; (2) Extravasation of monocytes from the blood vessels; (3) Macrophages are polarized to M1 type (pro-inflammatory); and (4) M2 type (anti-inflammatory) which causes induction of angiogenesis, anaerobic glycolysis, metastasis, invasion/migration, tumor proliferation and immune suppression; B: Representation of the Wnt signaling pathway in the activated and inactivated state and the role of Wnt signaling in different cancer hallmarks for tumor progression. Immune suppression: Tumor-associated macrophage (TAM) intrinsic β catenin signaling; Invasion/migration: TAM intrinsic β catenin signaling (Wnt 5a, Wnt 5b, Wnt 7b); Metastasis: Wnt 1, Wnt 2b, Wnt 3a; Tumor proliferation: TAM intrinsic β catenin signaling (Wnt 1, Wnt 3a, Wnt 5a, Wnt 5b); IFN: Interferon; TNF-α: Tumour necrosis factor-α; GM-CSF: Granulocyte macrophage colony stimulating factor; M-CSF: Macrophage colony-stimulating factor; LPS: Lipopolysaccharide; LRP: Lipoprotein receptor-related protein; GSK-3β: Glycogen synthase kinase-3β; CKIα: Casein kinase Iα; APC: Adenomatous polyposis coli; TCF: T cell factor; LEF: Lymphoid enhancer factor.

In the recent issue of the World Journal of Gastroenterology, an interesting article was published entitled ‘Calculus bovis inhibits M2 tumor-associated macrophage (TAM) polarization via Wnt/β-catenin pathway modulation to suppress liver cancer’[50]. Huang et al[50] described the anti-liver cancer mechanism of CB in which they depicted the inhibition of M2 TAM polarization via the Wnt pathway. In the pharmacodynamics study, they found an abundance of lithocholic acid and glycohyodeoxycholic acid in CB extract and CB-enriched serum, respectively. Upon construction of a “component-target-disease” network, 168 common anti-liver cancer targets were found, of which TNF and IL-6 were the central targets in Gene Ontology analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed significant pathways involved including the PI3K/AKT pathway, Wnt pathway, Rap1 pathway, and Ras pathway. GCA, taurodeoxycholic acid, glycohyodeoxycholic acid, hyodeoxycholic acid, and 7-ketolithocholic acid exhibited binding with the proteins of the β-catenin pathway. Also, transcriptomics revealed 359 upregulated and 461 downregulated genes. Altogether, this suggested the Wnt pathway as the key target of CB-mediated inhibition in cancer. Furthermore, stimulation of THP-1 cells with IL-13 and IL-4 showed the upregulation of CD206 with significant reductions in mRNA levels of CCL22, Arg-1, TGF-β2, and IL-10. In addition to this, CB-enriched serum impeded M2-TAM proliferation of liver cancer, diminished invasion and migration and the induction of apoptosis via downregulation of the Wnt pathway. In vivo results from the study further corroborated their findings which also showed the inhibition of M2-tumor associated macrophage polarization via the Wnt pathway. Therefore, the authors have depicted the use of Chinese herb viz. CB inhibited the polarization of M2 TAMs via downregulation of the Wnt pathway which could be a promising strategy to combat liver cancer. However, the article shows the results only from in vitro experiments and validation using in vivo models followed by confirmation in a cohort of liver cancer patients is required.

CONCLUSION

The investigation depicted the anti-neoplastic effects of CB by the inhibition of M2-polarized TAM differentiation in the TME. Comprehensive in vitro analysis showed the involvement of the Wnt signaling pathway as a molecular mechanism of anti-liver cancer activity of CB. While TCM herbs show promising anticancer properties, several significant challenges must be overcome. These challenges include inconsistent herb composition, a lack of standardized preparation methods, and potential interactions with conventional medications. More research is therefore required to establish standardized formulations, understand the precise mechanisms of action, and conduct thorough clinical trials to confirm their efficacy and safety. In addition, comparative studies are needed to identify which cancers could benefit most from CB treatment.

In conclusion, this study lays the foundation for the development of CB-derived antineoplastic therapeutic strategies to combat liver cancer. However, several key gaps in the literature need to be addressed to fully understand and optimize its therapeutic effects. First, the exact mechanisms of action of active compounds such as oleanolic acid, ergosterol, and ursolic acid remain largely unclear. Further studies are needed to elucidate their precise mechanisms, particularly concerning apoptosis and immune system interactions. Secondly, while studies indicate that cholic acid, TCA and GCA exhibit anti-inflammatory effects, their interactions with existing therapies need further validation through rigorous clinical trials. Lastly, the long-term effects and potential toxicity of CBS should be investigated to ensure safety for prolonged use in hemorrhagic liver diseases.