The results indi cated that the repair activity selleck chem Tubacin of APE1 probably participates in the melphalan resistance but is also due to elevated expression. To verify that the DNA lesions caused by melphalan were less prominent in melphalan resistant cells, an alkaline comet assay reflecting base or small patch DNA lesions was performed. In congruence with APE activity, DNA lesions induced by melphalan were repaired effectively in RPMI 8226 LR5 compared to the parental cells which partially explained the differ ential sensitivity to melphalan. The reduced DNA repair capacity of APE1 rendered more DNA single strand breaks in RPMI 8226 at 30 min post melphalan treat ment when compared to RPMI 8226 LR5. The overall DNA repair capacity of single stand breaks induced by melphalan Inhibitors,Modulators,Libraries further confirmed that the DNA repair activity of APE1 plays a critical role in melphalan resistance.
Taken together, our results indicated that APE1 DNA repair activity plays an important Inhibitors,Modulators,Libraries role in melphalan resistance. However, the DNA repair function mutant still increases the melphalan resistance in the APE1 knockdown background, which sug gested that acetylation modification of APE1 may also be involved in melphalan resistance. MDR1 expression is reduced in APE1 deficient MM cells We then analyzed the possible mechanism of APE1 acetylation mediated melphalan resistance in MM cells. Firstly, we detected the acetylation level of APE1 in mel phalan resistant MM cell lines and their wildtype coun terparts. As shown in Figure 6A, APE1 acetylation levels could be detected when APE1 was enriched by immuno precipitation.
Inhibitors,Modulators,Libraries After normalization with pan APE1, its Inhibitors,Modulators,Libraries acetylation level increased in the melphalan resistant MM cells RPMI 8226 LR5 and U266 LR6 in response to melphalan treatment. Due to the importance of MDR1 in drug resistance, we then measured differences in the expression of MDR1 between RPMI 8226 LR5 and its parental cell line. MDR1 is constitutively expressed to a higher level in melphalan resistant MM cells as shown in Figure 6A. Since a previous study reported that APE1 plays a critical role in regulation of MDR1 expres sion through a novel acetylation modification, we then postulated that APE1 could be involved in melphalan re sistance in MM cells by regulating MDR1 expression. First we observed that APE1 knockdown and overex pression in the RPMI 8226 cells manipulated the MDR1 protein expression level.
We then tested if the melphalan resistance induced by APE1 overexpres sion could be negated by knocking Inhibitors,Modulators,Libraries down MDR1 expres sion. gefitinib mechanism of action APE1 wildtype cDNA expression vector and a vector only control were transfected in parallel into RPMI 8226 cells. Both of these transfected cell lines were then transfected with siRNA specifically against Mdr1. Different groups of cells were then challenged with melphalan.