TO THE EDITOR
We conducted a comprehensive review of the study by Li et al[1], published in the World Journal of Gastrointestinal Oncology. Mou et al[2] indicated that icaritin, an 8-prenylated flavone, is derived from the traditional Chinese herb Epimedium Genus, which has historically been utilized as a tonic, aphrodisiac, and anti-rheumatic agent. Reyes-Hernández et al[3] noted that, following oral administration, icariin is hydrolyzed and deglycosylated by intestinal bacteria to produce icaritin, a metabolite with significant pharmacological potential. Zhang et al[4] reported that it demonstrates minimal toxicity in preclinical studies; However, Lu et al[5] observed that the application of this compound is constrained by its poor water solubility and low bioavailability. Gao and Zhang et al[6] reported that it exhibits a broad spectrum of non-neoplastic therapeutic effects, including osteoprotective, neuroprotective, cardiovascular protective, anti-cancer, anti-inflammatory, and immune-protective properties. Additionally, it demonstrates anti-tumor effects through the modulation of various signaling pathways. These pathways include the inhibition of the cell cycle, suppression of cell proliferation, invasion, and migration, regulation of autophagy, and induction of apoptosis and differentiation in cancers such as breast, lung, liver, prostate, and colorectal, as noted by Liu et al[7]. Colorectal cancer (CRC) represents a significant global health challenge. Siegel et al[8] indicated that epidemiological data show it ranks third in incidence among males and second among females, positioning it as a leading cause of cancer-related mortality worldwide. According to Zeng et al[9], the prognosis for patients with advanced CRC remains unfavorable, largely due to metastatic spread. Therefore, there is an urgent need to identify novel therapeutic compounds and molecular targets to combat CRC metastasis. Zhao et al[10] indicated that the Wnt/β-catenin signaling pathway is a well-established critical pathway associated with metastasis and invasion in CRC. Additionally, it plays a significant role in regulating epithelial-mesenchymal transition (EMT), a process that promotes tumor cell invasion and migration across various cancer types, as highlighted by Hu et al[11] and Lei et al[12]. According to Li et al[13], Zhang et al[14], and Li et al[15], icaritin has exhibited anti-tumor effects in CRC through multiple molecular mechanisms, including the inhibition of autophagy, suppression of cyclin-dependent kinase 2, induction of reactive oxygen species generation, and concurrent inhibition of key signaling proteins such as cyclin E, cyclin D1, and Bcl-2. Nevertheless, the effects of Icaritin on EMT and Wnt/β-catenin signaling in CRC have yet to be investigated.
Li et al[1] demonstrate that icaritin inhibits the proliferation, migration, and invasion of CRC cells, likely by modulating EMT and suppressing the Wnt/β-catenin pathway. This mechanism is systematically validated for the first time in both in vitro and in vivo models in this study. Furthermore, icaritin promotes apoptosis and inhibits cell migration and invasion while downregulating the expression of N-cadherin, Snail, β-catenin, matrix metalloproteinase-2, and matrix metalloproteinase-9. Simultaneously, it upregulates E-cadherin and adenomatous polyposis coli. These findings provide both experimental and theoretical support for the clinical application of icaritin in CRC, underscoring its potential as a promising candidate drug for CRC treatment.
This study could be strengthened by incorporating a broader range of supportive investigations to enhance the reliability of its findings. Morphology-based assessments would provide more intuitive observation of icaritin-induced EMT; however, the study lacks morphological examination of such EMT-related changes. The following supportive experiments could be employed to address this limitation: Analysis of changes in cell polarity via transmission electron microscopy reveals a loss of intercellular connections and suggests ultrastructural remodeling driven by EMT. Staining of intracellular F-actin with fluorescently labeled phalloidin, such as fluorescein isothiocyanate- or tetramethylrhodamine isothiocyanate-conjugated conjugates, clearly reveals a more dispersed distribution and altered morphology of the cytoskeleton. Alternatively, immunofluorescence staining using specific antibodies against epithelial and mesenchymal markers facilitates the examination of protein localization and expression intensity within cells. For example, E-cadherin is predominantly localized at the cell membrane in normal epithelial cells; however, its expression diminishes and its localization may change during EMT. In contrast, vimentin displays a fibrous distribution in mesenchymal cells, with increased expression and broader distribution during EMT. Refining these experimental methods would further strengthen the validity of the findings.
Furthermore, the mechanism by which icaritin exerts its inhibitory effect on CRC necessitates further elucidation. While the study confirmed that icaritin inhibits cell invasion and metastasis by modulating EMT and the Wnt/β-catenin signaling pathway in HCT116 and SW620 cells, it focused solely on a limited selection of Wnt/β-catenin pathway proteins (e.g., adenomatous polyposis coli, β-catenin) through Western blot analysis. This restricted examination fails to sufficiently establish the role of the Wnt/β-catenin signaling pathway. Validating the activation of the Wnt pathway requires a comprehensive, multi-level analytical approach that employs various experimental methods. Initially, in addition to assessing total protein expression, authors could evaluate β-catenin nuclear accumulation (e.g., immunofluorescence/confocal imaging and/or nuclear-cytoplasmic fractionation followed by western blot) and quantify canonical Wnt transcriptional output using T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter assays (e.g., wild-type TCF/LEF reporter/mutant TCF/LEF control reporter). Subsequently, Second, downstream Wnt target genes should be examined at the mRNA and/or protein level (e.g., axis inhibition protein 2, cellular myelocytomatosis oncogene, cyclin D1), together with β-catenin stability-related readouts (e.g., active/non-phosphorylated β-catenin and/or N-terminal phosphorylation associated with degradation) to better define the step targeted by icaritin. Finally, to establish a clear causal relationship and pinpoint where icaritin acts within the pathway, rescue experiments are warranted. If the phenotype is truly dependent on the Wnt/β-catenin axis, activating Wnt signaling (e.g., by adding Wnt3a or inhibiting glycogen synthase kinase 3 beta) or expressing constitutively active β-catenin should partially or completely reverse the anti-migratory/anti-invasive effects of icaritin. Conversely, β-catenin knockdown should phenocopy the effects of icaritin, and the combination of β-catenin knockdown with icaritin should show limited or no additional (additive) inhibition, thereby supporting the notion that both interventions converge on the same node within the pathway. Future bioinformatics analyses and the identification of molecular targets may facilitate the exploration of critical informational molecules and signaling pathways involved in the suppression of CRC metastasis and invasion by icaritin. These efforts will help uncover additional targets of icaritin and clarify its mechanisms related to migration and EMT in CRC. Furthermore, as noted by He and Gan[16], the Wnt/β-catenin pathway plays multiple roles in CRC. Its contributions to tumor initiation, proliferation, angiogenesis, and chemotherapy resistance during icaritin treatment merit further investigation.
Advancing the clinical application of icaritin in CRC necessitates the resolution of several key challenges. The mechanisms by which icaritin exerts its inhibitory effects on CRC are intricate and remain incompletely understood, warranting further investigation into its potential molecular targets. Additionally, icaritin is hindered by various limitations that compromise its anticancer efficacy, such as poor water solubility, low bioavailability, and insufficient oral absorption, which continue to obstruct its clinical application. It is imperative to explore more potent derivatives and diverse formulation strategies to enhance the solubility of poorly soluble compounds. As suggested by Szabó et al[17], this may involve employing surfactant compounds, co-solvents, emulsions, solid dispersions, cyclodextrins, and the preparation of salt forms. Furthermore, the majority of clinical studies examining icaritin in cancer therapy have concentrated on hepatocellular carcinoma, with five studies conducted, while only one study has investigated metastatic breast cancer. Regrettably, no data have been analyzed, reported, or published regarding CRC, as indicated by Reyes-Hernández et al[3]. There is an urgent need for additional clinical data and well-designed trials to assess the efficacy of icaritin in CRC. Furthermore, Wu et al[18] emphasized that the combination of natural products with other agents to overcome drug resistance mechanisms has emerged as a potent strategy for cancer treatment. Investigating the synergy between icaritin and chemotherapeutic agents - such as 5-fluorouracil, which targets the Wnt/β-catenin pathway in CRC, as noted by Cho et al[19], and doxorubicin - may clarify whether icaritin enhances effects via the Wnt/β-catenin pathway, thereby promoting the reversal of EMT and inhibiting CRC cell invasion and migration, while also preventing drug resistance. Such research could lead to novel clinical treatment strategies. Future investigations into the synergistic effects of icaritin in conjunction with other natural phytochemicals that operate through similar Wnt/β-catenin mechanisms in CRC, such as curcumin and crocin, as studied by Amerizadeh et al[20] and Marjaneh et al[21] may further expand the clinical applications of icaritin.
In summary, the study by Li et al[1] elucidates the potential mechanism by which icaritin inhibits CRC metastasis through the EMT transition and the Wnt/β-catenin signaling pathway. By innovatively linking the biological activity of icaritin to metastasis - a critical process in CRC progression - this research examines the role of icaritin in modulating EMT and the Wnt/β-catenin pathway, thereby providing a scientific foundation for its clinical application in CRC. However, additional experimental approaches are required to enhance the reliability of these findings. Moreover, deeper mechanistic insights necessitate further elucidation, and challenges related to the clinical translation of icaritin must be addressed to advance its application for patients with CRC.
