Anti-tumorigenic and anti-angiogenic effects of natural conifer Abies sibirica terpenoids in vivo and in vitro.

Aim: The natural terpenoid compound was explored in vitro and in vivo to investigate the anti-HCC properties.
Methods: For our study we used Abisilin ^® - a novel natural pharmacological terpenoid compound extracted and purified from coniferous Pinaceae trees. Anti-tumorigenic properties of different concentrations of Abisilin ^® were tested on murine hepatoma Hepa 1-6 cell lines. The analysis of proliferation and apoptosis was performed using immunofluorescence microscopy, FACS and qPCR. As an in vivo approach, we tested Abisilin ^® (400mg/kg/day, 14 days, orally) in xenograft mouse models of liver cancer and investigated tumor growth, proliferation, apoptosis and angiogenesis by means of Western blotting, immunofluorescence microscopy and qPCR.
Results: Application of Abisilin ^® for 24h at a dosage ranging from 0.03 to 0.045mg significantly reduced the number of viable Hepa 1-6 cells and induced apoptotic cell death with microscopic evidence of changes in cell morphology, and positive TUNEL, cleaved caspase 3 and Annexin V/Propidium Iodide (PI) stainings. Furthermore, treatment with Abisilin ^® strongly inhibited proliferation, impaired mitosis and prompted cell cycle arrest by down-regulation of the Cyclin D1, E1 and A2 expression levels. In Hepa 1-6 xenograft in vivo model, Abisilin ^® considerably decreased the xenograft tumor size and tumor volume. Consistently with in vitro Abisilin ^® administration elicited apoptosis and inhibit proliferation in the xenograft tumor. We also found that Abisilin ^® remarkably decrease microvessel density, diminished tumor angiogenesis and reduced expression of ICAM-1. Moreover, the expression of pAMPK, a cellular energy sensor, was up-regulated after Abisilin ^® application.
Conclusions: Anti-proliferative, pro-apoptotic activity and anti-angiogenic potential of natural conifer terpenoids might turn these compounds into an attractive drug candidate for combination therapy against liver cancer.
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