br Xia et al reported that exosomes released by
Xia et al. reported that exosomes released by A549 Methoxy-X04 exposed to DDP (cis-diamminedichloroplatinum, cisplatin) could communicate with other cells and influence the resistance of these cells to DDP . Since aspirin could change the secretory characteristics of exosome in A549 cells, we further investigated whether aspirin augments DDP-in-duced cell apoptosis under hypoxic conditions. As expected, hypoxia reduced DDP sensitivity compared with that of the normoxia group, and an enhanced population of apoptotic cells was found in A549 cells treated with aspirin and DDP in hypoxia (Fig. 7A, B). Inhibition of
exosome secretion by GW4869, yielded similar results. In addition, we also used the method of co-incubation of exosomes and other cells to detect the effect of exosomes on the efficacy of cisplatin. The results of cell viability assay showed that hypoxia-induced exosomes significantly attenuated the inhibitory effect of cisplatin on the proliferation of A549 cells, while the inhibition effect of cisplatin was significantly reduced in the aspirin-treated exosomes group (Fig. 7C). These data suggest that aspirin and DDP exert a synergistic effect on apoptosis under hypoxic conditions in A549 cells, at least in part, through the exosome pathway.
Hypoxia is a common feature of the TME of solid tumors due to their rapid expansion and complex vasculature; it is associated with ag-gressive tumorigenic properties and poor clinical outcomes . Hy-poxic induction of gene expression occurs mainly through HIF-1α
Fig. 6. Correlation between exosome and proliferation and stemness of A549 cells under hypoxia. (A) A549 cells were treated with 10 μM GW4869 or 2.5 mM Aspirinfor 72 h, and cell viability was detected by CCK-8 assay. (B, C) The second generation of ALDH1+ cells was treated with different conditions of stimulation for 72 h, the proportion of ALDH1+ cells in cell cultures was analyzed by FACS. Representative examples are displayed. Each bar indicates the mean ± SD of n = 3 experiments; * indicates P < 0.05, ** indicates P < 0.01.
accumulation. HIF-1α has been reported to induce the transcriptional activation of COX-2 in A549 cells under hypoxic conditions [33,34]. Activation of the COX-2/PGE2 pathway plays an important role in cancer cell survival and tumor angiogenesis [9,34]. Moreover, COX-2 is frequently overexpressed in NSCLC and associated with poor prognosis in NSCLC patients . These findings suggest that COX-2/PGE2 may be an important target of NSCLC therapy.
Aspirin, an NSAID, is also known as a non-selective COX inhibitor [35,36]. The drug was recently reported to inhibit epithelial-me-senchymal transition and migration of highly invasive K-ras-expressing NSCLC cells by targeting Slug . However, the role of aspirin in tumorigenesis under hypoxic conditions remains unknown. Direct in-hibition of COX-2 is generally accepted as the main pathway by which aspirin inhibits cancer development . We thus analyzed the function of the COX-2/PGE2 pathway in the anti-tumor effect of aspirin during hypoxia. As expected, aspirin inhibited tumorigenesis and stemness under hypoxic conditions via HIF-1α/COX-2/PGE2 signaling in NSCLC cells. This finding is consistent with a recent study reporting that COX-2 maintains the stemness of nasopharyngeal carcinoma cell lines . Collectively, our findings indicate that aspirin has therapeutic potential for suppressing lung cancer development and stemness.
Emerging evidence indicates that communication within the com-ponents of the TME could promote tumorigenesis through the release of exosomes [38,39]. Protein estimation coupled with exosome quantifi-cation assay suggests that hypoxia significantly increases exosome protein concentrations and numbers of exosomes in conditioned medium. Hypoxia also resulted in a remarkable increase in exosomal HIF-1α and COX-2 protein levels. These findings are consistent with a
recent report demonstrating that hypoxia promotes the release of exo-somes in breast cancer cells and that this process may be mediated by HIF-1α . Interestingly, we found that aspirin inhibited the hypoxia-enhanced release of exosomes, which could be partly mediated by the HIF-1α/COX-2 pathway. Exosomes secreted by aspirin-treated cells inhibited hypoxia-enhanced cell proliferation, migration, and angio-genesis in A549 cells in vitro. The data indicate that hypoxic A549 cells release large amounts of exosomes into their TME to promote their own survival, migration, and tube formation and that this process could be partially abrogated by aspirin. Besides increases in exosome number, we also demonstrated that miR-135b and miR-210 are elevated in hy-poxic exosomes [24,27] Given the roles of exosomal miR-135b and miR-210 in promoting angiogenesis and metastasis in response to hy-poxia [24–26], these exosomal miRNAs may be potential therapeutic targets during hypoxia. Here, we showed that aspirin reduces the ex-pression of exosomal miR-135b and miR-210 under hypoxic conditions. In short, aspirin may inhibit hypoxic enhancement of exosome release via the HIF-1α/COX-2 pathway and target exosomal miR-135b and miR-210 to inhibit cell proliferation, migration, and angiogenesis in NSCLC cells. Our experimental data further suggest that the synergistic effect of aspirin and cisplatin on lung cancer cells may be due to changes in exosome function. These findings provide novel insights into the mechanism of aspirin-inhibited tumorigenesis during hypoxia.