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Desialylation of MM overcomes bortezomib resistance in vivo
aggressive MM mouse model, which employs xenotrans- plantation of MM cells that have been enriched for E- selectin ligands.14 To this end, we used the pan-sialyltrans- ferase inhibitor 3Fax-Neu5Ac, which had been previously shown to efficiently block sialylation in leukemic cells.24
In a preliminary dose-finding in vivo study, we observed that 3Fax-Neu5Ac decreased sialylation in various tissues, including cells of the immune system. At its effective
A
dose, 25 mg/kg, 3Fax-Neu5Ac induced edema in the peri- toneal cavity of mice suggesting that desialylation of the glomerulus could lead to dose-limiting toxicity, as previ- ously reported.27 To overcome 3Fax-Neu5Ac-induced kid- ney toxicity and to examine the role of sialylation specifi- cally in MM, we treated the E-selectin enriched MM1S cell line, MM1SHeca452, with 3Fax-Neu5Ac before inoculation, an approach that has been successfully used to uncover a crit-
B
C
Figure 4. 3Fax-Neu5Ac treatment has a minimal impact on stroma-mediat- ed bortezomib resistance in MM1SHeca452 cell line. MM1SHeca452 cells were treated with 300 μM 3Fax-Neu5Ac or dimethyl sulfoxide (DMSO) [vehicle control (CTRL)] for seven days. After treatment, cells were seeded onto 80% confluent layer of HS5 expressing GFP (A), patient- derived bone marrow stromal cells (BMSC) (B) and BMEC-60 stained with Tag-it VioletTM (C) or plastic. BMEC-60 were also stimulated with 10 ng/mL TNFα for 4 hours (h) to induce activation. Cells were co-cul- tured for 24 h and then treated with 5 nM bortezomib for a further 24 h. After incubation, cells were collected and cell death was examined by Annexin V-APC and PI staining. One- way ANOVA was used to determine statistical significance with Dunnett’s multiple comparison post-hoc testing. *P<0.05; **P<0.01; ***P<0.001; ns: non-significant.
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