Figure 1B and C show both APE1 mRNA and protein levels were higher in the melphalan resistant cell lines sug gesting that APE1 is a melphalan responsive gene. To check this hypothesis we challenged the cells with 15 uM melphalan for 1 hour then measured APE1 ex pression differences in both wildtype selleck chemicals llc RPMI 8226 and Inhibitors,Modulators,Libraries RPMI 8226 LR5 cells. As shown in Figure 2A and B, APE1 mRNA expression was elevated after melphalan treatment as early as 3 hours while protein level was ele vated at 18 hours after treatment. The peak of APE1 protein elevation was at 24 hours after melphalan treat ment and the mRNA peak was at 12 hours. The signifi cant elevation of APE1 expression was observed in a dose dependent fashion at 24 hours post melphalan treatment.

On the other hand, the APE1 level, which is already high in RPMI 8226 LR5 cells, Inhibitors,Modulators,Libraries failed to show a significant increase until high dose treatment of melphalan. These correlative data suggested that APE1 Inhibitors,Modulators,Libraries could play a role in a melphalan induced cellular response and consequently promote re sistance to melphalan. Manipulation of APE1 affects cell resistance to melphalan To further confirm the role of APE1 in melphalan resist ance, we utilized RNAi and vector based overexpression strategies to manipulate cellular APE1 expression in wildtype RPMI 8226 cells. The changes in drug resist ance were then observed in cells with exogenously al tered APE1 expression. RNAi was performed using adenovirus Inhibitors,Modulators,Libraries previously engineered by our lab and its effi cacy was confirmed by Western blot.

Both RNAi and overexpression effectively altered total cellular APE1 protein levels at 48 hours after transduction according to the Western blot shown in Figure 3A. Noteworthy, since the B cell origin Inhibitors,Modulators,Libraries of both parental MM cell lines, we found that the APE1 knockdown rendered Sorafenib Tosylate mw no signifi cant growth inhibition under untreated conditions, which agrees with previous reports. The melpha lan resistance was then tested by CCK 8 assay in the groups with different APE1 expression levels. The cell killing effects by melphalan were measured at 24, 48 and 72 hours after 15 uM melphalan treatment. Figure 3B clearly indicates that APE1 deficiency sensitized RPMI 8226 cells to melphalan, meanwhile, overexpression of APE1 rendered 8226 cells with enhanced resistance to melphalan. In addition, we also tested if APE1 inhibition in RPMI 8226 LR5 cells could restore the sensitivity to melphalan. At 48 hours post transfection, APE1 protein levels were effectively downregulated as shown in Figure 3C. At 48 hours following melphalan treatment, CCK 8 assays were performed to measure the cell viabil ity in different groups.