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Several treatment strategies are used for multiple myeloma (MM), because of the high heterogeneity of MM, it still remains an incurable disease and it gives different clinical response against the therapeutic agents and drugs. Bortezomib, proteasome inhibitor, is an anticancer agent used for the treatment of multiple myeloma while methylstat is a demethylase inhibitor having anticancer potential. Until now, the effects of methylstat have been reported in breast and oesophageal cancer types. However, there is no study on multiple myeloma cells (MM). 

Cytotoxic effects of bortezomib and methylstat on U266 and ARH77 cells were demonstrated by MTT cell proliferation assay. To understand the apoptotic effects of these agents, loss of mitochondrial membrane potential and changes in caspase-3 enzyme activity were investigated by using JC-1 dye based-method (Cayman Chemicals, USA) and caspase-3 colorimetric assay kit (BioVision Research Products, USA) while phosphatidylserine localization was investigated by Annexin V assay. Cell cycle analysis in response to Bortezomib and Methylstat alone or in their combination were measured by flow cytometry. Changes in expression profiles of 84 genes underlying apoptosis and cell cycle control, in response to Methylstat were determined by Human Cancer Pathway Finder RT² Profiler PCR Array System (SABiosciences Corporation, USA).

There were dose- and time- dependent decreases in cell proliferation in response to bortezomib and methylstat based on the results of MTT assay. IC50 values were calculated from cell proliferation plots and found to be 41 nM and 19 nM for U266 and ARH77 cells in respons to bortezomib, respectively while they were 2.2 µM and 4.2 µM for U266 and ARH77 cells in response to methylstat, respectively. On the other hand, combination studies showed that IC50 values decreased as compared to bortezomib and methylstat alone and were calculated as 0.5 nM and 0.6 nM for U266 and ARH77 cells, respectively. To evaluate the apoptotic effects of agents, JC-1, caspase-3 and Annexin-V analysis were performed. Combination of bortezomib with methylstat showed more effect on apoptosis as compared to any agent alone and untreated control group.  Also, cell cycle analysis was performed to detect the DNA contents of cells. Combination of bortezomib and methylstat arrested cells at the S phase. The fraction of cells in S phase increased accordingly.  Previous data demonstrated that combination of agents caused apoptosis and it could be cell cycle-dependent. Human Cancer Pathway Finder RT² Profiler PCR Array System results of this study had shown that 1.1 µM and 2.2 µM methylstat treatment caused upregulation of FASLG, NGFR, TNF, TNFRS10B and TNFRS1B apoptosis- triggering genes in U266 cells and BCL2L11, CSP7, TNFRSF21 and 2.1 µM and 4.2 µM methylstat treatment caused TNFSF8 apoptosis- triggering genes in ARH77 cells in a dose-dependent manner. Furthermore, there were significant decreases in the expression levels of AKT1, AVEN, BAG1 BCL2L2 and RELA anti-apoptotic genes in U266 cells and NFKB1 anti-apoptotic gene in ARH77 cells in response to increasing concentrations of methylstat.

In conclusion, all results showed that the effects of bortezomib in combination with methylstat on U266 and ARH77 MM cells had significant decreases in proliferation and triggered apoptosis. Lots of genes and pathway in the cell were affected by methylstat treatment so methylstat might be used as candidate agent for the treatment of MM after in vivo analyses.