Consequently, treatment with HDACIs would alter the FPGS promoter acetylation status and increase FPGS gene transcription and intracellular accumulation of long-chain MTX-PGs based on in vitro assessments of acute lymphoid leukemia cell lines.32 Vorinostat (molecular excess weight: 264) is a small-molecule inhibitor of most human class I and class II HDACs. and DHFR in 2 human PCNSL-derived cell lines (HKBML and TK) and a human Burkitt lymphoma cell collection (TL-1). Combination treatments were created using 4 HDACIs: panobinostat, vorinostat, sodium butyrate, and valproic acid. The expression of DHFR was examined as well as ratios of FPGS/GGH expression. The combined effects of MTX plus HDACIs were evaluated using a cell viability assay, mass spectroscopy imaging, and subcutaneous and intracranial xenograft models. Results HDACIs upregulated the ratio of FPGS/GGH expression resulting in increased polyglutamylation of MTX, but also downregulated expression of the target molecule of MTX: DHFR. The combination of MTX and vorinostat decreased cell viability in vitro ( .05) and tumor volumes in a subcutaneous model ( .0001), and prolonged survival in an intracranial model ( .01), relative to controls. Conclusion HDACIs enhanced the therapeutic effect of MTX through increased polyglutamylation of MTX and concomitant downregulation of DHFR expression. test, based on the mean standard deviation. Survival analyses were performed using the KaplanCMeier method and the log-rank test. Differences were considered statistically significant at values of less than .05. All analyses were performed using IBM SPSS software (version 19; IBM Corp.). Results Effects of MTX Treatment and LV Rescue Were Related to Polyglutamylation in Lymphoma Cell Lines The cytotoxic effects of MTX on HKBML, TL-1, and TK cells were examined using the in vitro CellTiter-Glo assay. The IC50 values for MTX were 71.1 nM for HKBML cells, 21.7 nM for TL-1 cells, and 25.8 nM for TK cells (Determine 1A). When we used 100 nM of MTX, the cytotoxic effects Bergamottin reached almost maximum in all cell lines. There were a few differences in the cell viability Bergamottin between 100 and 1000 nM of MTX usage (data not shown). Cell viabilities of HKBML, TL-1, and TK cells in the maximal cytotoxic effects of MTX were about 30%, 20%, and 5%, respectively. Over 90% cytotoxicity was reached only in TK cells. Open in a separate window Physique 1. Effects of methotrexate (MTX) treatment and leucovorin (LV) rescue were related to the degree of polyglutamylation in lymphoma cell lines. (A) Viability assays using cell lines after incubation with MTX (CellTiter-Glo). Each cell collection was analyzed after 72 h of incubation. (b) Cells were treated with MTX for 24 h, followed by the addition of LV. Cell viability was assessed 48 h later. We defined EC50 as the concentration of LV that recovered 50% cell viability. Data are shown as mean value SD from 3 impartial experiments. * .01, compared with MTX. (C) Immunoblotting for folypolyglutamate synthetase (FPGS), -glutamyl hydrolase (GGH), and dihydrofolate reductase (DHFR) in the different cell lines. The internal control was -tubulin. The relative expression of FPGS/GGH represents the ratio of FPGS/-tubulin and GGH/-tubulin calculated by densitometry. The FPGS/GGH ratio of HKBML is usually adjusted to 1 1. Data are shown as mean value SD from 3 impartial experiments; * .05, ** .01. The EC50 was defined as the LV concentration to Bergamottin recover 50% cell viability, which was found to be 17.6 ng/mL for HKBML cells, 41.8 ng/mL for TL-1 cells, and 125.4 ng/mL for TK cells (Determine 1B). These results indicated that HKBML cells were more easily rescued by LV, relative to the other cell lines. We compared the expressions of FPGS, GGH, and DHFR among all cell lines using Western blotting c-Raf (Physique 1C). The results revealed that FPGS expression was highest in TK cells and that GGH expression was highest in HKBML cells. The expression level of DHFR was highest in TL-1 cells and was not different between HKBML and TK cells. The FPGS/GGH ratio was highest in TK cells and least Bergamottin expensive in HKBML cells, which was consistent with EC50 values for LV. Therefore, the response to MTX and LV rescue appears to be associated with the FPGS/GGH ratio, which nearly reflected the extent of MTX polyglutamylation. Effect of HDACIs on Lymphoma Cell Lines We examined the effects of HDACIs on lymphoma cell lines. In addition to NaBu, we used panobinostat, vorinostat, and VPA because these drugs are approved by the FDA for other diseases. The IC50 values for each drug were evaluated in the lymphoma cell lines (Table 1; Supplementary Physique S1). As the IC50 values for panobinostat and vorinostat were lower than the values for NaBu and VPA, panobinostat and.