Excessive production of ROS causes oxidative stress and damage to various cellular elements, like lipids, enzymes, proteins, cell membranes, and DNA in the living cells. It eventually promotes tumor development and progression by intervening in crucial cellular procedures involving apoptosis, proliferation, migration, and resistance (20). According to our results, exogenic SP, the natural ligand for the NK1 receptor, induced ROS production in breast cancer cells, suggesting the SP/NK1R signaling is linked to BC pathogenesis by oxidative stress. In line with our results, some investigations reported that ROS production elevated in response to SP in the immune-inflammatory cells (21, 22), sensory neurons (23), and epithelial cells of the gastric and respiratory tract (24, 25). For example, SP-activated NK1R enhances the hemorrhagic lesion's development of the stomach barrier via increasing the cytotoxic ROS generation in the gastric epithelial cells (25). SP operates the neutrophil NADPH oxidase enzyme, one of the primary origins of intracellular ROS, related to NK1R to produce cytotoxic ROS, resulting in the hyperactive bladder (26–28).
Many investigations have been concentrated on the antitumoral influences of aprepitant on various cancer cells (29, 30). Our findings indicated that aprepitant exerts antitumoral effects, at least partly, by reversing the SP induced-ROS production.
In line with our results, a study has suggested that aprepitant could reduce cisplatin-induced ROS generation in PC12 of BC cells treated with aprepitant (15 µM) just 2 hours before cisplatin treatment (31). Another study that evaluated the aprepitant effect on ROS production proposed that the administration of aprepitant (35 µM) in U87 glioblastoma cells can significantly reduce ROS levels after 24 hours (12). In a similar investigation on U87 glioblastoma cells, ROS level remarkably was reduced as an effect of aprepitant (15 µM) treatment for 24 hours (32).
In contrast with these findings, there are some investigations reported that aprepitant positively regulates ROS production in K562 and HL60 myeloid leukemia tumor cells, treated with aprepitant (24 µM and 19µM, sequentially) for 3 hours (33) and TNBC (triple-negative BC) cell line, MDA-MB 231, pretreated with aprepitant for 2 hours (34). It seems aprepitant probably has the dual effect of regulating ROS production in tumor cells considering applied dose and exposure time. In addition, this contrast might be the differences in SP and aprepitant dosages used, oxidative capacity of the cancer cells, time of exposure to drugs, and the diverse redox conducting signaling pathways used via various cell types. These results, which accompany our previous findings, suggest that aprepitant might be able to promote antitumoral effects through suppressing ROS production (35). Although these studies have distinguished the role of SP on ROS generation, further verification in further studies is indispensable to clarify the exact molecular mechanism implied in the effects of SP in ROS induction in BC cells.
To evaluate the probable SP and aprepitant effects on cellular redox state, we tried to determine the gene and protein expression of Trx, one of the essential components of the enzymatic antioxidant defense system, upon aprepitant exposure of MCF-7 cells. Our findings revealed that the NK1R repression by aprepitant induced the gene and protein expression of Trx. In a study that operated on the U87 glioblastoma, similar to our findings, SP inhibited the expression of the Trx gene, and conversely, the aprepitant induced Trx gene expression significantly (32).
miRNAs participate in conducting and regulating their related target genes expression and play a crucial part in the occurrence and progression of several cancers (36–38). The microRNAs prediction database, TargetScan 7.2, anticipated that miR-325-3p could straight target Trx. MiR-325-3p is reported to promote cancer cell proliferation, metastasis, and EMT in vitro (39). For intense, it has been shown that an increased miR-325-3p gene expression is linked to inhibition of invasion and proliferation of non-small cell lung cancer by differently targeting (39, 40). It is also reported miR-325-3p overexpression inhibits glioma (41) and bladder cancer cells growth and metastasis (42).
Moreover, an investigation on hepatocellular carcinoma proved that miR-325-3p can prevent cell proliferation and lead to cell apoptosis induction (43). Recently, studies revealed that miR-325-3p increases in cancer, such as BC tissues and cell lines (44). To estimate the possible effect of SP/NK1R on miR-325-3p, we assessed its expression and found that miR-325-3p gene expression was elevated in the MCF-7 cell line. We also found that the miR-325-3p gene expression was following the Trx expression. In the current study, we observed that SP/NK1R could have a dual manner on the expression of miR-325-3p.
The current study explored the miR-325-3p and its target gene, Trx relation, and the SP and aprepitant effects on them in the BC for the first time. We found that the NK1R antagonism, aprepitant, remarkably prevents the Trx system and miR-325-3p alteration mediated by SP.