br Fig Aspirin inhibits the hypoxia mediated stemness of
Fig. 3. Aspirin inhibits the hypoxia-mediated stemness of ALDH-positive cells. (A) Representative flow cytometric plots of the analysis of ALDH1 enzymatic activity in A549 K 252a under normoxic or hypoxic conditions. (B) The second generation of ALDH1+ cells was treated with different conditions of stimulation for 48 or 72 h, and cell viability was detected by CCK-8 assay. (C, D) The proportion of ALDH1+ cells in cell cultures treated under different conditions of stimulation was analyzed by FACS. Representative examples are displayed. (E) OCT4, HIF-1α, and SOX-2 protein levels were determined by Western blot, with GAPDH serving as the loading control. (F) The concentration of PGE2 secreted by ALDH+ cells was determined by ELISA. Each bar indicates the mean ± SD of n = 3 experiments; ns indicates non-significant, * indicates P < 0.05, ** indicates P < 0.01.
3.2. Aspirin inhibits hypoxia-mediated lung cancer cell stemness
Hypoxia is well known to induce the stemness phenotype of various cancer cells, including pancreatic cancer, glioblastoma, and lung cancer [6,19,20]. To investigate the effect of aspirin on the stemness pheno-type, we evaluated the levels of stemness-related markers in human non-small-cell lung cancer cell line A549 during hypoxic conditions. As shown in Fig. 2A–C, mRNA levels of SOX2, OCT4, and aldehyde de-hydrogenase1 (ALDH1) significantly increased under hypoxia and de-creased under pre-treatment with aspirin. Similar results were observed in the protein levels of the corresponding genes, as determined by Western blot (Fig. 2G). To explore the signaling pathway involved in these changes, we examined the expression levels of HIF-1α and COX-2; among these factors, the latter is a known target of aspirin. Hypoxia up-regulated HIF-1α and COX-2 mRNA (P < 0.01) and protein levels when compared with the corresponding levels observed in the control group (Fig. 2D, E, and G). Treatment with aspirin resulted in decreased expression of HIF-1α and COX-2 under hypoxic conditions (Fig. 2D, E, and G). The hypoxia-enhanced secretion of PGE2 into the culture medium was also decreased by aspirin (Fig. 2F). Taken together, the results indicate that aspirin may inhibit hypoxia-mediated stemness via inactivation of HIF-1α/COX-2/PGE2 signaling.
3.3. Aspirin inhibits the stemness of ALDH-positive cells
Over the past few decades, increasing evidence has supported the role of ALDH1 as a reliable marker for the isolation of CSCs [21,22]. Jiang et al. reported that isolated lung cancer cells with high ALDH1 activity display CSC-like features . As hypoxic conditions are re-sponsible for maintaining the stemness of lung CSCs, we investigated the inhibitory effect of aspirin in ALDH+ cells. Aldefluor assay followed by FACS analysis showed 0.02% ALDH1+ cells in control cells and 2.07% ALDH1+ cells in hypoxic A549 cells (Fig. 3A). Therefore, we separated subpopulations in hypoxic A549 cells according to ALDH1 using FACS cell sorting to enrich in vitro cultures of CSCs. ALDH+ A549 cells may undergo asymmetrical division to generate heterogeneous subpopulations. To eliminate this impact, second- and third-generation ALDH+ cells were used in the subsequent experiments. CCK-8 assays illustrated that hypoxia promotes the proliferation of ALDH+ cells whereas aspirin remarkably inhibits cell growth at 48 and 72 h (Fig. 3B). FACS analysis showed hypoxia-induced ALDH+ cell enrich-ment and impairment of this enrichment by aspirin in a dose-dependent manner (Fig. 3C,D). Western blot consistently revealed that ALDH+ cells express high levels of the stemness-related markers SOX2 and OCT4 under hypoxic conditions but dose-dependent decreases upon
Fig. 4. Aspirin affects the characteristics of exosomes secreted by A549 cells under hypoxic conditions. (A) Transmission electron microscopy of exosomes isolated by the ExoQuick-TC kit. (B) Levels of exosomal proteins CD63 and CD81 and the endoplasmic reticulum marker calnexin were determined by Western blot. The data reported are the representative results of three independent experiments. (C) The number of exosomes from each sample was quantitated by the EXOCET exosome quantification assay kit. (D) Levels of exosomal proteins HIF-1α and COX-2 were determined by Western blot, with CD63 serving as the internal control. The data reported are the representative results of three independent experiments. (E) Exosomal miR-16, miR-135b, and miR-210 were determined by qRT–PCR. Aspirin is used at a concentration of 10 mM. Data were normalized to exogenous cel-miR-39. Each bar represents the mean ± SD of n = 3 experiments; ** indicates P < 0.01.
aspirin treatment (Fig. 3E). Western blot coupled with ELISA (Fig. 3F) indicated that the hypoxia-mediated activation of HIF-1α/COX-2/PGE2 signaling could be attenuated by aspirin; the second and third genera-tions of ALDH+ cells showed similar results (Supplementary Fig. 1). These findings suggest that aspirin inhibits the hypoxia-mediated maintenance of stemness in ALDH+ cells.