Aptamer TY04 Inhibits Multiple Myeloma Cell Growth Via Cell Cycle Arrest

Multiple myeloma (MM), also known as plasma cell myeloma, is characterized by accumulation of clonal plasma cells in the bone marrow and overproduction of monoclonal immunoglobulin (Ig) in the blood or urine. MM accounts for approximately 10% of all hematologic malignancies. Despite recent advances in the understanding and treatment of this disease, MM remains an incurable disease in the vast majority. With conventional chemotherapy, the 5-year median survival rate for MM patients is approximately 25%. Aptamers are single-stranded RNA or DNA sequences that bind to target molecules with high affinity and specificity. Compared with antibodies, aptamers have unique advantages including easy chemical synthesis and modification, low toxicity, lack of immunogenicity, and rapid tissue penetration, Based on these advantages, aptamers show great potential for therapeutic application. The aptamer TY04 is a single-stranded DNA (ssDNA) generated by a method named cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), We found TY04 strongly inhibited the growth of multiple myeloma cell lines including MM1.S, NCI-H929, KM3 and OPM2,The concentration of TY04 to inhibit 50% cell growth (IC50) on MM1.S was 3.89 μM. In contrast, TY04 had no effect on the growth of non-tumor cell lines — immortal B lymphoblastoid cell lines. Next, we used MM1.S cell line as the model to study the mechanism of TY04 anti- multiple myeloma. Flow cytometry analysis showed that TY04 with the sequence specifically bind to MM1.S cells when compared with unselected ssDNA library control. To investigate whether the target molecules of TY04 are membrane proteins on cell surface, MM1.S cells were treated with trypsin and proteinase k for 2 or 10 minutes before incubation with TY04. The result revealed that TY04 lost partly recognition ability on treated cells, indicating that the target molecules were most likely membrane proteins. Furthermore, we evaluated the cell cycle distribution of MM1.S after TY04 treatment. We found that TY04 significantly caused cell-cycle arrest in G2/M phase. The percentage of G2/M phase cells increased from 30.1±1.56 to 53.2±6.36. To identify the underlying molecular mechanism, G2/M-related proteins were assayed by flow cytometry. Following TY04 treatment, a concomitant inhibition of ERK1/2, cyclin B, CDK1 and γ-tubulin expression occurred. Meanwhile, human cell cycle PCR array was used to analyze the expression of 84 genes key to cell cycle regulation in TY04-treated MM1.S cells. Our results indicated that aptamer TY04 decreased the genes expression of CCNB1, CCNB2, BIRC5, BRCA1 and CCNH, which were involved in the progress of G2/M phase. All these results are significant in that they provide a framework for further exploring the use of TY04 as a novel anti-multiple myeloma agent.