RNAi-mediated knockdown of aldehyde dehydrogenase class-1A1 and class-3A1 is specific and reveals that each contributes equally to the resistance against 4-hydroperoxycyclophosphamide

Purpose: Aldehyde dehydrogenases class-1A1 (ALDH1A1) and class-3A1 (ALDH3A1) have been associated with resistance to cyclophosphamide (CP) and its derivatives. We have previously reported the downregulation of these enzymes by all-trans retinoic acid (ATRA). Methods: In this study, we used siRNA duplexes as well as retrovirally expressed siRNA to knockdown one or both enzymes together in A549 lung cancer cell line in order to investigate the role of each one in mediating the resistance and the effect of the addition of ATRA. Results: The results show that significant and specific knockdown of each enzyme can be achieved and that each one contributes similarly to cell resistance to 4-hydroperoxycyclophosphamide (4-HC), an active derivative of CP. Added effects were seen when both enzymes were inhibited. The addition of ATRA also exhibited additional inhibitory effects on ALDH activity and increased 4-HC toxicity when added to single siRNA aimed at one of the enzymes. On the other hand, ATRA had minimal and insignificant additional inhibitory effects on ALDH enzyme activity when added to a combination of siRNAs against both enzymes, but still increased 4-HC toxicity beyond that seen with RNAi-mediated inhibition of both enzymes together. Conclusions: We conclude that both enzymes, ALDH1A1 and ALDH3A1 will need to be blocked in order to achieve the highest sensitivity to 4-HC. Furthermore, ATRA increases 4-HC toxicity even when added to a combination of siRNAs against both enzymes, thus suggesting additional mechanisms by which ATRA can increase drug toxicity.