IntroductionHepatocellular carcinoma (HCC) remains a major global health burden, ranking as the sixth most prevalent cancer and the third leading cause of cancer-related mortality worldwide1. Egypt exhibits a particularly high incidence of HCC, ranking third in Africa and 15th globally2. Doxorubicin (DOX), an anthracycline antibiotic, is commonly used in HCC management due to its significant therapeutic potential; however, cardiotoxicity and drug resistance remain as major limitations3. Its mechanism of action involves inhibiting topoisomerase II, disrupting deoxyribonucleic acid (DNA) replication, and generating free radicals, thereby inducing cellular damage4. Due to limitations of the conventional chemotherapeutic agents, there is increasing interest in investigating alternative therapies to enhance chemotherapy efficacy and mitigate toxicity5. For instance, Licochalcone B has been shown to ameliorate liver cancer by targeting apoptotic genes and DNA repair systems6, while natural agents like camel milk and bee honey have demonstrated efficacy in regulating profibrotic cytokines in carbon tetrachloride (CCl4)-induced cirrhosis7. These studies underscore the validity of exploring natural antioxidants.A key pathway of interest is the canonical Wnt/β-catenin pathway, which is essential in hepatic development, normal liver function, and tissue homeostasis; its dysregulation is implicated in various liver diseases and cancer8. Under normal conditions, in the absence of a Wnt ligand, cytoplasmic β-catenin levels are kept low through phosphorylation and destruction by the destruction complex, resulting in the repression of Wnt target genes9. Conversely, activation of Wnt signaling pathway via binding of Wnt ligands to their receptors disrupts the destruction complex. This leads to cytoplasmic β-catenin accumulation, and its translocation to the nucleus, where it functions as a transcription activator of target genes essential for cell proliferation and survival, such as cyclin D1, cellular myelocytomatosis (c-Myc) and multidrug resistance protein-1 (MDR1)10.Astaxanthin (ATX), a xanthophyll carotenoid and red fat-soluble pigment, exhibits potent biological antioxidant activity compared to other carotenoids. It is naturally present in various marine organisms and microorganisms, such as shrimp, krill, salmon and Haematococcus pluvialis (H. pluvialis), a green microalga rich in ATX content11. Its chemical structure comprises a six-membered ring with two terminal rings joined by conjugated double bonds, which not only determine its color but also govern its biological function12. ATX exhibits a diverse range of biological activities, including potent antioxidant activity attributed to its distinctive molecular structure, containing hydroxyl and keto moieties on each ionone ring13. It also possesses anti-lipid peroxidation activity, protecting the cell membrane by overlaying its terminal polar groups with the polar regions of the cell membrane, while fitting its strongly hydrophobic conjugated polyene structure to the inner non‑polar region. Thus, ATX spans biological membranes, maintaining the membrane structure, inhibiting lipid peroxidation, and acting as an antioxidant14.Additionally, ATX demonstrates anticancer properties through various mechanisms, such as anti-proliferation, modulation of apoptosis, anti-oxidation, cell cycle arrest, and cell growth inhibition15,16. ATX has been shown to increase the levels of glycogen synthase kinase-3β (GSK3β) and suppress the nuclear transfer of β-catenin. It also modulates protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) phosphorylation, consequently inhibiting the nuclear factor kappa B (NF-κB) and Wnt/β-catenin pathways17. Therefore, this study aims to investigate the possible therapeutic and/or adjuvant effects of ATX in rat model of HCC.ResultsExpression of β-catenin, GSK3β and AFP levelsSignificant alterations in β-catenin expression levels were observed among the experimental groups. Specifically, untreated HCC rats exhibited a considerable elevation in β-catenin expression, showing an approximately 3.5-fold increase compared to the control group (Fig. 1a). Conversely, rats treated with ATX, DOX, or combination therapy showed reductions in β-catenin expression levels by 31%, 29%, and 58%, respectively, relative to the untreated HCC group. Moreover, HCC rats treated with combination therapy exhibited a notably decreased expression of β-catenin compared to ATX- and DOX-treated rats by approximately 38%, and 40%, respectively.Regarding GSK3β expression, untreated HCC rats exhibited decreased levels of GSK3β protein compared to the control group (0.71-fold decrease). However, HCC rats treated with either ATX or combination therapy showed a remarkable increase in GSK3β levels compared to untreated rats, by approximately 33.6% and 41%, respectively (Fig. 1b).Additionally, alpha-fetoprotein (AFP), a glycoprotein serving as a key biomarker for HCC diagnosis and monitoring, demonstrated elevated levels in untreated HCC rats compared to the control group, reaching a 2-fold increase (Fig. 1c). Conversely, HCC rats treated with either ATX or DOX exhibited significant reduction in AFP level, decreasing by approximately 46% and 37%, respectively, compared to untreated rats. HCC rats treated with combination therapy showed a remarkable decrease in AFP levels, reducing them by 49% compared to the untreated group.Fig. 1The alternative text for this image may have been generated using AI.Full size imageEffect of ATX and DOX on hepatic Wnt pathway proteins and AFP levels. Protein levels of (a) β-catenin, (b) GSK3β, and (c) AFP were assessed using ELISA technique. Data are expressed as mean ± standard deviation (SD) (N = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test, with significance set at (P