Anti-Myeloma Activity of Modified Galectin-9 through JNK and p38 MAPK Pathways.

Abstract 952

Galectins constitute a family of animal lectins that show affinity for β-galactosides and share conserved amino acid sequences in their carbohydrate recognition domains (CRDs). Galectin-9 is a tandem-repeat type galectin with two CRDs and has been shown to be crucially involved in a variety cellular functions, such as cell adhesion, cell proliferation, apoptosis and mRNA splicing. Recent studies have indentified anti-cancer properties of galectin-9 against several cancerous diseases through the induction of apoptosis and the prevention of metastasis. In this study, we investigated for the first time the anti-multiple myeloma (MM) activity of a recombinant mutant form of human galectin-9 (hGal9) both in vitro and in vivo, and also examined the potential utility of hGal9 for treatment-resistant MM. hGal9 was synthesized by the Galpharma Research Institute (Kagawa, Japan). While the natural form of galectin-9 having a long linker peptide is a substrate for proteolysis, the deletion of the entire linker region renders hGal9 highly stable against proteolysis. In vitro, hGal9 inhibited the cell proliferation of four myeloma cell lines examined (KMS-12-BM, AMO-1, NCI-H929, IM9), including a bortezomib-low sensitive IM9 subcell line, with IC₅₀ between 75.1 and 280.0 nM. This growth inhibitory effect was mediated by the induction of apoptosis as was demonstrated by the increased number of Annexin V-positive cells with the activation of caspase-8, -9, and -3 in hGal9-treated cells. β-galactoside binding activity is essential in the anti-myeloma activity of hGal9, as the addition of lactose completely abrogated hGal9-mediated growth inhibitory effect on myeloma cell lines and the degree of cell surface binding affinity of hGal9 tends to correlate with IC₅₀ of hGal9 in specific cell lines. To determine the molecular pathway responsible for the anti-myeloma activity of hGal9, we used RNA microarray technology to analyze the changes in gene expression patterns by hGal9. Two MM cell lines, IM9 and KMS-12-BM, were analyzed, and the genes showing more than 2.0-fold changes in their expression level after 6h of hGal9 treatment in both cell lines were assessed. Sixteen genes were upregulated both in KMS-12-BM and IM9, and we focused on c-jun (2.64-fold in IM9 and 6.90-fold in KMS-12-BM) and junD (2.14-fold in IM9 and 3.03-fold in KMS-12-BM), major effector molecules downstream of JNK and p38 MAPK, which are known to be essential for cell survival and death regulation of MM. The upregulation of c-Jun and JunD proteins by hGal9 was also confirmed with Western blotting. We then focused on the relationship between the anti-MM effect of hGal9 and the cross-talk networks of JNK and p38 MAPK signaling pathways. JNK and p38 MPAK phosphorylation by hGal9 were evident in both cell lines, and hGal9 also induced H2AX phosphorylation in myeloma cell lines, suggesting that JNK-H2AX and/or p38-H2AX signaling pathways are involved in the apoptotic induction by hGal9 in myeloma cells. Importantly, the blockade of either the JNK by JNK inhibitor VIII or p38 MAPK pathway by SB203580 partially eliminates the cytotoxic effect of hGal9 on myeloma cells. Taken together, our findings suggest that the anti-myeloma activity of hGal9 at least partly mediates the activation of the JNK and p38 MAPK pathways. hGal9 also induced cell death in patient-derived CD138-positive myeloma cells from ten patients, some with poor risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. Collectively, our study suggest that hGal9 is a potential candidate for a new therapeutic approach for MM that may overcome resistance to conventional chemotherapy.