Pulmonary fibrosis is characterized by fibroblast proliferation and extracellular matrix remodelling, leading to respiratory insufficiency. The mechanisms underlying this progressive and devastating disease remain unclear. Conditions that can impair the function of the endoplasmic reticulum (ER) cause accumulation of unfolded or misfolded proteins, resulting in ER stress and activation of the unfolded protein response (UPR). ER stress has been implicated in many conditions including cancer, diabetes, obesity, and inflammation. It is also involved in lung fibrosis, through myofibroblastic differentiation of fibroblasts; however, the precise role of ER stress in lung fibrosis is unknown. The current study aimed to investigate the underlying mechanisms of ER stress inhibitors in the treatment of bleomycin-induced lung fibrosis. We demonstrated that bleomycin can activate ER stress associated proteins, including GRP78, CHOP, and ATF-4, both in vitro and in vivo. PI3K/AKT acts upstream of ER stress to affect lung fibroblast proliferation, resulting in bleomycin-induced pulmonary fibrosis. Treatment with ER stress inhibitors or a PI3K inhibitor caused a reduction in fibroblast proliferation and improved pulmonary function. The relationship between PI3K/AKT/mTOR and ER stress in pulmonary fibrosis, and the application of PI3K inhibitors and ER stress inhibitors in the treatment of pulmonary fibrosis require further investigation.
Tumors are influenced by a microenvironment rich in inflammatory cytokines, growth factors and chemokines, which may promote tumor growth. Interleukin-6 (IL-6) is a multifunctional cytokine and known as a regulator of immune and inflammation responses. IL-6 has also been reported to be associated with tumor progression and chemoresistance in different types of cancers. In our study, we demonstrated that IL-6 enriches the properties of lung cancer stem-like cells in A549 lung cancer cells cultured in spheroid medium. IL-6 also promotes sphere formation and stem-like properties of A549 cells by enhancing cell proliferation. Methylation-specific polymerase chain reaction (PCR) was performed and revealed that IL-6 increased methylation of p53 and p21 in A549 cancer cells. Western blot analysis and quantitative real-time PCR demonstrated that IL-6 increased the expression of DNA methyltransferase 1 (DNMT1) in A549 cells cultured in spheroid medium, but not the expression of DNMT3a or DNMT3b. Knockdown of DNMT1 eliminated IL-6-mediated hypermethylation of cell cycle regulators and enrichment of lung cancer stem-like properties. In conclusion, our study, for the first time, shows that the IL-6/JAK2/STAT3 pathway upregulates DNMT1 and enhances cancer initiation and lung cancer stem cell (CSC) proliferation by downregulation of p53 and p21 resulting from DNA hypermethylation. Upon blockage of the IL-6/JAK2/STAT3 pathway and inhibition of DNMT1, the proliferation of lung CSCs was reduced and their formation of spheres and ability to initiate tumor growth were decreased. These data suggest that targeting of the IL-6/JAK2/STAT3 signaling pathway and DNMT1 may become important strategies for treating lung cancer.Key words: IL-6/JAK2/STAT3 pathway, DNMT1, lung cancer stem cell, proliferation Abbreviations: BCA: bicinchoninic acid; bFGF: basic fibroblastic growth factor; BrdU: bromodeoxyuridine; CDK: cyclin-dependent kinase; CSCs: cancer stem cells; DAPI: 4 0 ,6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; DNMT1: DNA methyltransferase 1; EGFR: epidermal growth factor receptor; EMT: epithelial mesenchymal transition; FACS: fluorescence-activated cell sorting; FBS: fetal bovine serum; HDAC1: histone deacetylase 1; IL-6: interleukin-6; JAK2: Janus kinase 2; M-PER: mammalian protein extraction reagent; miRNA: microRNA; MS-PCR: methylation-specific polymerase chain reaction; NF-kB: nuclear factor-kappa B; NOD/ SCID: nonobese diabetic/severe combined immunodeficient; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PI: propidium iodide; PKB: protein kinase B; PVDF: polyvinylidene difluoride; RT-PCR: real-time polymerase chain reaction; SD: standard deviation; SDS: sodium dodecyl sulfate; shRNA: small hairpin RNA; STAT3: signal transducer and activator of transcription 3; TBST: Trisbuffered saline and Tween 20; TIMP-3: tissue inhibitor of metalloproteinase 3; TUNEL: terminal deoxynucleotidyl transferase dUTP
Epithelial-to-mesenchymal transition (EMT) is associated with tumor metastasis and tumorigenesis in lung cancer stem-like cells (CSCs). However, the exact mechanism underlying this is not clear. We used microarray analysis to identify candidate genes responsible for EMT in spheroid and monolayer cultures of lung cancer cells. We found increased expression of a variety of adhesion molecules in CSCs. One of these molecules, Collagen XVII (Col XVII), was demonstrated to be required for maintenance of EMT phenotypes and metastasis ability in lung CSCs. We showed that Col XVII stabilized laminin-5 to activate the FAK/AKT/GSK3β pathway, thereby suppressing Snail ubiquitination-degradation. The function of Col XVII was mainly dependent on shedding by ADAM9 and ADAM10. Patients who underwent surgical resection for lung cancer, and displayed overexpression of both Col XVII and laminin-5, had the worst prognosis of all expression types. Moreover, blockage of the Col XVII/laminin-5 pathway reduced the EMT phenotypes of lung CSCs in vitro and decreased the potential of lung metastasis in vivo. Our findings suggested that targeting Col XVII and laminin-5 could be novel therapeutic strategies for treating lung cancer patients, and warrant further investigation.
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