A zebrafish HCT116 xenograft model to predict anandamide outcomes on colorectal cancer.

Colon cancer is one of the leading causes of death worldwide. In recent years, cannabinoids have been extensively studied for their potential anticancer effects and symptom management. Several in vitro studies reported anandamide's (AEA) ability to block cancer cell proliferation and migration, but evidence from in vivo studies is still lacking. Thus, in this study, the effects of AEA exposure in zebrafish embryos transplanted with HCT116 cells were evaluated. Totally, 48 hpf xenografts were exposed to 10 nM AEA, 10 nM AM251, one of the cannabinoid 1 receptor (CB1) antagonist/inverse agonists, and to AEA + AM251, to verify the specific effect of AEA treatment. AEA efficacy was evaluated by confocal microscopy, which demonstrated that these xenografts presented a smaller tumor size, reduced tumor angiogenesis, and lacked micrometastasis formation. To gain deeper evidence into AEA action, microscopic observations were completed by molecular analyses. RNA seq performed on zebrafish transcriptome reported the downregulation of genes involved in cell proliferation, angiogenesis, and the immune system. Conversely, HCT116 cell transcripts resulted not affected by AEA treatment. In vitro HCT116 culture, in fact, confirmed that AEA exposure did not affect cell proliferation and viability, thus suggesting that the reduced tumor size mainly depends on direct effects on the fish rather than on the transplanted cancer cells. AEA reduced cell proliferation and tumor angiogenesis, as suggested by socs3 and pcnp mRNAs and Vegfc protein levels, and exerted anti-inflammatory activity, as indicated by the reduction of il-11a, mhc1uba, and csf3b mRNA. Of note, are the results obtained in groups exposed to AM251, which presence nullifies AEA's beneficial effects. In conclusion, this study promotes the efficacy of AEA in personalized cancer therapy, as suggested by its ability to drive tumor growth and metastasis, and strongly supports the use of zebrafish xenograft as an emerging model platform for cancer studies.

epg5 knockout leads to the impairment of reproductive success and courtship behaviour in a zebrafish model of autophagy-related diseases.

BACKGROUND: Dysregulation of the autophagic flux is linked to a wide array of human diseases, and recent findings highlighted the central role of autophagy in reproduction, as well as an association between impairment of autophagy and behavioural disorders. Here we deepened on the possible multilevel link between impairment of the autophagic processes and reproduction at both the physiological and the behavioural level in a zebrafish mutant model. METHODS: Using a KO epg5 zebrafish line we analysed male breeding success, fertility rate, offspring survival, ejaculate quality, sperm and testes morphology, and courtship behaviour. To this aim physiological, histological, ultrastructural and behavioural analyses on epg5+/+ and mutant epg5-/- males coupled to WT females were applied. RESULTS: We observed an impairment of male reproductive performance in mutant epg5-/- males that showed a lower breeding success with a reduced mean number of eggs spawned by their WT female partners. The spermatogenesis and the ability to produce fertilising ejaculates were not drastically impaired in our mutant males, whereas we observed a reduction of their courtship behaviour that might contribute to explain their lower overall reproductive success. CONCLUSION: Collectively our findings corroborate the hypothesis of a multilevel link between the autophagic process and reproduction. Moreover, by giving a first glimpse on behavioural disorders associated to epg5 KO in model zebrafish, our results open the way to more extensive behavioural analyses, also beyond the reproductive events, that might serve as new tools for the molecular screening of autophagy-related multisystemic and neurodegenerative diseases.