Background: Carcinoid tumors of the gastrointestinal tract often metastasize to the liver and are associated with debilitating symptoms and a poor quality of life. In the human gastrointestinal carcinoid cell line BON, the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) has been shown to inhibit growth and decrease the secretion of hormones. Lithium is an inhibitor of the glycogen synthase kinase 3β (GSK-3β) pathway. The combination of distinct mechanisms of action could be an effective treatment modality with lower toxicity. We hypothesized that more significant growth inhibition could be achieved with combination therapy of both drugs.
Methods: BON cells were treated with varying combinations of 0-20 µM SBHA and 0-20 mM lithium chloride for up to 96 hours. Western analysis was used to measure neuroendocrine tumor markers achaete-scute complex-like 1 (ASCL1) and chromogranin A (CgA). Growth was measured by a methylthiazolyldiphenyl-tetrazolium bromide cellular proliferation assay, and Western analysis was used to determine the mechanism of growth regulation and the effects on the Notch-1 and GSK-3β pathways.
Results: A dose-dependent decrease in ASCL1 and CgA with the combination of 20 mM lithium and 5-20 µM SBHA was observed after 48 hours; this was more pronounced at 96 hours. SBHA increased the amount of active Notch-1 protein. Lithium was associated with phosphorylation of GSK-3β, demonstrating inhibition of the pathway. Growth was inhibited equally by 20 µM SBHA and the combination of 10 µM SBHA with 20 mM lithium after 96 hours. The levels of both p21 and p27 increased, suggesting that growth inhibition occurs through cell cycle arrest.
Conclusions: Treatment of BON cells with SBHA and lithium chloride suppresses neuroendocrine markers ASCL1 and CgA while upregulating Notch-1 and inhibiting GSK-3β. This combination reduces growth through cell cycle arrest, and these outcomes were attained with lower doses in combination than treatment with either compound alone. With equal efficacy and theoretically decreased toxicity, combination therapy may be a viable therapeutic approach for gastrointestinal carcinoid tumors.