Exploring the Effect of Glucosamine Hydrochloride on Liver Cancer Using Zebrafish Liver Cancer Model

Objective: To explore the effects of Agaricus bisporu-derived glucosamine hydrochloride (GAH) on hepatocellular carcinoma (HCC). Methods: A zebrafish liver cancer and vascular double transgenic model, Tg (flk:mCherry;krasG12V) was induced using a 60 mg/mL doxycycline hydrochloride solution. Larvae were traced and observed for the development of liver cancer from 5 d to 10 d using laser confocal microscopy. Different concentrations of GAH were added during Dox induction, larvae in Control group (Ctr), modeling group (Dox), Dox+0.1% GAH group and Dox+0.3% GAH group (Dox) were analyzed under laser confocal microscopy to observe the effect of GAH on liver cancer and tumor vascular growth. The effect of GAH on the mRNA and protein expression of HCC and vascular-related regulatory factors (prmt5, tiam1, kat5, vegfaa, vegfr2, tgf-β1) was analyzed via qPCR and ELISA assays. Finally, the correlations between tgf-β1, prmt5, tiam1, kat5, vegfaa and vegfr2 were validated through TIMER2 online bioinformatics analysis tool. Results: Continuous induction of Dox facilitated the emergence of HCC. Compared with the Dox model group, the growth and invasion of HCC were significantly inhibited after GAH treatment (P<0.001), and the vascular disorder of HCC was greatly improved (P<0.001). GAH intervention markedly ameliorated vascular dysregulation associated with liver cancer (P<0.001). qPCR and ELISA assays revealed that GAH could effectively suppress the mRNA and protein levels of genes associated with HCC and its vasculature (P<0.05). The significance of tgf-β1 in the expression of genes related to HCC and vascular pathways was proved using the TIMER2 database. Conclusion: GAH would potentially inhibit tumor growth and invasiveness by regulating the TGF-β signaling pathway, thereby influencing the expression of genes related to HCC and inhibiting the development of HCC vasculature.