Galectin-9, a soluble β-galactoside-binding animal lectin, evokes apoptosis in various human cancer cell lines. The galectin-9 antitumor effect against hepatocellular carcinoma (HCC) is, however, unknown. We investigated whether galectin-9 suppresses HCC growth in vitro and in vivo. We assessed the antitumor effect of galectin-9 on HCC cells by conducting WST-8 assay in vitro and xenograft model analysis in vivo. Galectin-9-induced apoptosis was evaluated by FACS and ELISA in vitro and by TUNEL stain in vivo. Cell cycle alteration was profiled by FACS. Caspases were profiled by colorimetry. MicroRNAs related to the galectin-9 antitumor effects were determined using microarrays, and their antitumor effect was confirmed in a transfection study in vitro. The expression levels of the target proteins of the miRNAs extracted above were analyzed by western blot analysis. To summarize the results, galectin-9 inhibited the growth of the HCC cell lines HLE and Li-7 in vitro and Li-7 in vivo inducing apoptosis. Cell cycle turnover was not arrested in HLE and Li-7 cells in vitro. miR-1246 was similarly extracted both in vitro and in vivo, which sensitized Li-7 cells to apoptosis when transfected into the cells. DYRK1A, a target protein of miR-1246 was downregulated in Li-7 cells. Caspase-9 was upregulated in Li-7 cells in vitro and in vivo. In conclusion, galectin-9 inhibited the growth of HCC cells by apoptosis, but not cell cycle arrest, in vitro and in vivo. miR-1246 mediated signals of galectin-9, possibly through miR-1246-DYRK1A-caspase-9 axis. Galectin-9 might be a candidate agent for HCC chemotherapy.

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Abstract. Metformin is a commonly used oral antihyperglycemic agent of the biguanide family. Recent studies suggest that metformin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of metformin in several types of cancers, including hepatocellular carcinoma (HCC), has not been elucidated. The goal of the present study was to evaluate the effects of metformin on HCC cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin in vitro. We used the cell lines Alex, HLE and Huh7, and normal hepatocytes to study the effects of metformin on human HCC cells. In an in vivo study, athymic nude mice bearing xenograft tumors were treated with metformin or left untreated. Tumor growth was recorded after 4 weeks, and the expression of cell cycle-related proteins was determined. Metformin inhibited the proliferation of Alex, HLE and Huh7 cells in vitro and in vivo. Metformin blocked the cell cycle in G0/G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, cyclin E and cyclin-dependent kinase 4 (Cdk4). In addition, microRNA (miRNA) expression was markedly altered by the treatment with metformin in vitro and in vivo. In addition, various miRNAs induced by metformin also may contribute to the suppression of tumor growth. Our results demonstrate that metformin inhibits the growth of HCC, possibly by inducing G1 cell cycle arrest through the alteration of microRNAs.

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Dysregulation of apoptosis is a major hallmark in cancer biology that might equip tumors with a higher malignant potential and chemoresistance. The anti-cancer activities of lectin, defined as a carbohydrate-binding protein that is not an enzyme or antibody, have been investigated for over a century. Recently, galectin-9, which has two distinct carbohydrate recognition domains connected by a linker peptide, was noted to induce apoptosis in thymocytes and immune cells. The apoptosis of these cells contributes to the development and regulation of acquired immunity. Furthermore, human recombinant galectin-9, hG9NC (null), which lacks an entire region of the linker peptide, was designed to resist proteolysis. The hG9NC (null) has demonstrated anti-cancer activities, including inducing apoptosis in hematological, dermatological and gastrointestinal malignancies. In this review, the molecular characteristics, history and apoptosis-inducing potential of galectin-9 are described.