A. M. Waters1, J. E. Stewart1, V. R. Atigadda1, D. D. Muccio1, C. J. Grubbs1, E. A. Beierle1 1University Of Alabama,Birmingham, Alabama, USA
Introduction: Hepatoblastoma, a malignant tumor that arises from immature liver cells, is the most commonly diagnosed liver tumor in children. The incidence of hepatoblastoma is increasing but survival for children with advanced, metastatic or recurrent disease remains dismal at less than 50%. Therefore, innovative therapeutic strategies are needed to treat this disease. A novel retinoid, 9-cis-UAB30 (UAB30), has been developed that has significantly less toxicity than other retinoids. We hypothesized that treatment with UAB30 would induce cell cycle arrest, inhibit cellular migration and invasion, and lead to apoptosis and cell death in hepatoblastoma cells in vitro and impede tumor growth in vivo.
Methods: The human hepatoblastoma cell line HuH-6 was utilized. Cell cycle analysis was completed with flow cytometry. Migration and invasion assays were performed with scratch assays and Transwell plates. AlamarBlue® and trypan blue exclusion was used to measure cell survival and proliferation, respectively. Apoptosis was detected via Western blotting for PARP cleavage and caspase 3 activity was detected by a colorimetric assay kit. HuH-6 hepatoblastoma cells (2.5 x 106) were injected into the right flank of athymic nude mice. Mice were fed standard chow or UAB30-treated chow and tumor volumes were measured twice weekly with calipers. Animals were euthanized when parameters mandated by IACUC policy were reached. Student’s t-test was used to compare data between groups, with p ≤ 0.05 considered significant.
Results: The following results were noted with increasing concentrations of UAB30: a) cell cycle arrest with a significant increase in the percentage of cells in G1 and a decrease in S phases; b) a significant decrease in cellular migration and invasion; c) a significant decrease in cellular proliferation; and d) a significant decrease in cell survival. Further, increased PARP cleavage and caspase 3 activity confirmed that cell death was due to apoptosis. Additionally, after four weeks of treatment in the in vivo study, tumor size of the UAB30 treated mice injected with HuH-6 cells were significantly smaller than the controls (275 ± 402 mm3 vs. 1035 ± 164 mm3, treated vs. control, p<0.0001).
Conclusion: The synthetic retinoid, 9-cis-UAB30, had a significant impact upon the HuH-6 human hepatoblastoma cell line resulting in cell cycle arrest, decreased proliferation, migration and invasion, and increased apoptosis in vitro as well as inhibition of tumor growth in vivo. These results suggest a potential therapeutic role for UAB30 in hepatoblastoma treatment.