In this study, we show that exposure of human lung cancer A549 cells to cisplatin (cis-diamminedichloroplatinum, CDDP) promotes production of nitric oxide (NO) through generation of reactive oxygen species (ROS) and resulting upregulation of inducible NO synthase (iNOS). The incubation of the cells with a NO donor, diethylenetriamine NONOate, not only reduced the CDDP-induced cell death and apoptotic alterations (induction of CCAAT-enhancer-binding protein homologous protein and caspase-3 activation), but also elevated proteolytic activity of 26S proteasome, suggesting that the activation of proteasome function contributes to the reduction of CDDP sensitivity by NO. Monitoring expression levels of six aldo-keto reductases (AKRs) (1A1, 1B1, 1B10, 1C1, 1C2, and 1C3) during the treatment with the NO donor and subsequent CDDP sensitivity test using the specific inhibitors also proposed that upregulation of AKR1B10 by NO is a key process for acquiring the CDDP resistance in A549 cells. Treatment with CDDP and NO increased amounts of nitrotyrosine protein adducts, indicative of peroxynitrite formation, and promoted the induction of AKR1B10, inferring a relationship between peroxynitrite formation and the enzyme upregulation in the cells. The treatment with CDDP or a ROS-related lipid aldehyde, 4-hydroxy-2-nonenal, facilitated the iNOS upregulation, which was restored by increasing the AKR1B10 expression. In contrast, the facilitation of NO production by CDDP treatment was hardly observed in AKR1B10-overexpressing A549 cells and established CDDP-resistant cancer cells (A549, LoVo, and PC3). Collectively, these results suggest the NO functions as a key regulator controlling AKR1B10 expression and 26S proteasome function leading to gain of the CDDP resistance.
Summary Cisplatin (CDDP) is widely prescribed for the treatment of various cancers including bladder cancers, whereas its clinical use for breast cancer chemotherapy is restricted owing to easy acquisition of the chemoresistance. Here, we established a highly CDDP-resistant variant of human breast cancer MCF7 cells and found that procuring the resistance aberrantly elevates the expression of aldo-keto reductase (AKR) 1C3. Additionally, MCF7 cell sensitivity to CDDP was decreased and increased by overexpression and knockdown, respectively, of AKR1C3, clearly inferring that the enzyme plays a crucial role in acquiring the CDDP resistance. The CDDP-resistant cells suppressed the formation of cytotoxic reactive aldehydes by CDDP treatment, and the suppressive effects were almost completely abolished by pretreating with AKR1C3 inhibitor. The resistant cells also exhibited the elevated glutathione amount and 26S proteasomal proteolytic activities, and their CDDP sensitivity was significantly augmented by pretreatment with an inhibitor of glutathione synthesis or proteasomal proteolysis. Moreover, the combined treatment with inhibitors of AKR1C3, glutathione synthesis, and/or proteasomal proteolysis potently overcame the CDDP resistance and docetaxel cross-resistance. Taken together, our results suggest that the combination of inhibitors of AKR1C3, glutathione synthesis, and/or proteasomal proteolysis is effective as an adjuvant therapy to enhance CDDP sensitivity of breast cancer cells.
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