br Introduction br Lung cancer is a very common
Lung cancer is a very common cancer and the leading cause of cancer-related deaths worldwide [1,2]. Despite recent advances in clinical practice, including target therapies, however, the 5-year sur-vival rate of lung cancer remains at approximately 16%. One of the main reasons behind the poor prognosis of this cancer type is its re-sistance to various treatments . A subpopulation of cancer AM-251 with treatment-resistant potential is characterized by the abilities of self-renewal, differentiation, and metastasis, and cells of this subpopulation are called cancer stem cells (CSCs) . Developing specific therapies targeting CSCs is important to improve the clinical outcomes of lung cancer.
The tumor microenvironment (TME) of a solid tumor is composed of
cancer cells, stromal cells, blood vessels, and immune cells ; it has been implicated in the regulation of cell proliferation, invasion, and metastasis and contributes to the outcome of cancer therapy. Solid tu-mors feature hypoxic microenvironments due to their rapid expansion and irregular blood flow. Accumulating evidence suggests that hypoxia is associated with the invasive and metastatic potential of cancer cells, as well as their resistance to radio- and chemotherapies, all of which lead to poor clinical outcomes [4,5]. Hypoxia up-regulates the expres-sion of hypoxia-inducible factor-1α (HIF-1α), which, in turn, regulates various biological signals, including stem cell markers (e.g., SOX2 and OCT4) . Hypoxia has consistently been reported to promote cancer cell stemness and drug resistance in lung cancer . HIF-1α also up-regulates cyclooxygenase-2 (COX-2) during hypoxia, thereby leading to the increased abundance of its enzymatic product prostaglandin E2
(PGE2). Previous studies have reported that COX-2/PGE2 signaling plays crucial roles in cancer cell proliferation, apoptosis, angiogenesis, and stemness [8,9].
Emerging evidence has revealed that cancer-derived exosomes participate in the biological mechanism of the TME . Exosomes, which are small vesicles with diameters measuring 30–100 nm, have been shown to carry microRNAs (miRNAs), mRNAs, DNA fragments, and proteins [10,11]. A recent study demonstrated that exosomes se-creted under hypoxia enhance the invasiveness and stemness of cancer cells and induce TME recruitment and reprogramming in prostate cancer . However, the role of exosomes in non-small cell lung cancer (NSCLC) cells during hypoxia remains unclear.
Aspirin, a non-selective COX inhibitor, has been widely used as a nonsteroidal anti-inflammatory drug (NSAID) for over 100 years. Besides its well-known anti-inflammatory effects, aspirin has been re-ported to inhibit tumorigenesis in various tissues . Epidemiological and clinical studies have illustrated that regular aspirin consumption decreases the risk of NSCLC [14–17], which indicates that aspirin could be a promising agent in the treatment of this cancer. However, the underlying mechanism by which aspirin exerts its anti-tumor role in NSCLC has yet to be determined. The priming effect of aspirin on lymphoma was found to be associated with the altered constitution of TME . We thus hypothesize that aspirin may inhibit tumorigenesis in NSCLC by inducing TME changes under hypoxic conditions.
In this study, we demonstrate that aspirin inhibits the stemness of NSCLC A549 cells and weakens the malignant function of exosomes under hypoxic conditions. These effects could be responsible for the anti-tumor effect of the drug.
2. Materials and methods
2.1. Cell culture and treatment
Human lung adenocarcinoma cell line (A549) and human umbilical vein endothelial cells (HUVEC) were purchased from Cell Resource Center, Peking Union Medical College. Cells were cultured in RMPI-1640 supplemented with 10% Fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), 100U/ml penicillin and 100 μg/ml strepto-mycin. Cell culture was maintained at 37℃ in a humidified atmosphere with 5% CO2. For treatments, cells pre-treated with vehicle or different doses of aspirin (Sigma-Aldrich, St Louis, MO, USA) for five min, fol-lowed by a hypoxic process with 5% CO2, 2% O2, and 93% N2 in a 37℃ incubator.
2.2. Cell proliferation assay
A549 cells were seeded in 96-well plates at a density of 5 × 103 cells per well for24 h before treatment. After 12 h, 24 h, 48 h and 72 h of cultivation, cell proliferation was measured by Cell Counting Kit-8 (CCK-8) system (Beyotime, Haimen, China) according to the manu-facturer’s instruction. In brief, 10 μL of CCK-8 solution was added to each well and incubated at 37℃ for 1 h. The absorbance was measured at 450 nm with a microplate spectrophotometer (BioTek, USA). There were triplicates for each group, and the experiments were repeated at least three times.