Inhibition of Molecular Signaling in Huh-7 Cells by AM3: A Novel Chemotherapeutic Agent for Hepatocellular Carcinoma

Background: According to a review of recent literature, no previous studies have reported the dose-dependent selective inhibition of the antiproliferative activity using colony and sphere formation assays and immunoblotting in human hepatoma cells in response to doxorubicin and mitoxantrone structural analogs such as AM3. Objective: We evaluated the anticancer activity of mitoxantrone (MIT) structural analogs 1,5-bis({2- [(2-hydroxyethyl) amino]ethyl}-amino)-anthracene-9,10-dione (AM3) in human hepatoma cells (Huh-7). Methods: In this paper, we synthesized AM3 through the nucleophilic amino substitution of 1,5- dichloroanthraquinone with the corresponding dichloride groups under microwave-accelerated heating. The structural characteristics of AM3 were analyzed through ultraviolet–visible spectroscopy and nuclear magnetic resonance. In vitro activity of AM3 was measured using the dose-dependent selective inhibition of the antiproliferative activity using colony and sphere formation assays and immunoblotting in Huh-7. Results: The antiproliferative activity of AM3 was determined using IC50 values as 2.03 and 1.70 μM for hepatocellular carcinoma cell lines Huh-7 and SK-Hep-1 cells, respectively. In addition, colony formation assay of Huh-7 cells revealed that AM3 significantly suppressed the mean colony formation rate from 99.9 % to 2.5 %, and growth inhibition rate of sphere cells was significant, in which 5.0 μM of AM3 inhibited up to 28.5 % cell growth in the Huh-7 sphere cells. Immunoblotting confirmed the overexpression of CD44, COX-2, p-Akt, and Akt. Conclusion: Thus, AM3 is a novel therapeutic agent for suppressing cancer stemness and inflammation signaling in Huh-7 cells.