Data Availability StatementAll datasets presented in this study are included in the article/supplementary material. sensitized NSCLC cells to celecoxib-induced apoptosis by activating caspase-9, -8, -3, and -7, upregulating the pro-apoptotic proteins Bad and Bax, and downregulating the antiapoptotic proteins Bcl-xl and Bcl-2. Moreover, the superior anticancer effect of combined therapy was also due to suppression of Raf-MEK-ERK cascades and PI3K-AKT signaling, which is conducive to overcoming drug resistance. In addition, either celecoxib alone or Y-33075 dihydrochloride in combination with metformin suppressed NSCLC cell migration and invasion by inhibiting FAK, N-cadherin, and matrix metalloproteinase-9 activities. Together, our study provided a rational combination strategy with a low dosage of celecoxib and metformin for preclinical cancer application. experiments showed that combination therapy inhibits tumor growth in A549 xenograft-bearing nude mice more effectively than metformin and celecoxib alone. This study provides an effective combination treatment strategy for patients with NSCLC. Materials and Methods Materials A549 and H1299 cells were purchased from the American Type Culture Collection (ATCC, Philadelphia, PA, USA). Antibodies used for WB are listed as following: -actin (Abgent, San Diego, USA), N-Cadherin, p-FAK, FAK, MMP-9, Cleaved PARP, Cleaved caspase-9, Cleaved caspase-8, Cleaved caspase-7, Cleaved caspase-3, Bcl-2, Bcl-xl, Bad, Bax, Cyto-c, -H2AX, p53, p21, Cdc25A, p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, p-PI3P, PI3P, p-AKT, AKT (Cell Signaling Technology, Beverly, MA, USA), p-ATM, p-CHK2, CDK2 (Abcam Inc., Cambridge, MA). Celecoxib(98%,HPLC), metformin(purity: 99.9%), and pifithrin- were purchased from Sigma (St. Louis, USA). Cell Culture A549 and H1299 cells were grown in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Rabbit polyclonal to ZNF317 Invitrogen, Carlsbad, CA, USA). All cells were cultured in a humidified CO2 incubator at 37C. Cell Viability Assays Cells were digested and counted by an Y-33075 dihydrochloride automated cell counter (Invitrogen, Carlsbad, CA, USA), and 100 l of 5,000 cells were added to each well in a 96-well plate. Cells were incubated for 12?h and cultured in the incubator to form monolayers. The 96-well plate was changed to cell culture medium with different drug concentrations (metformin: 4 mM, 8 mM, 10 mM, 12 mM, 16 mM; celecoxib: 4 M, 8 M, 10 M, 12 M, 16 M), and then incubated for an additional 24 or 48?h. Cell viability was determined by the CCK-8 kit (Beyotime Inst Biotech, China). The absorbance was measured at 450 nm by a TECAN Safire Fluorescence Absorbance and Luminescence Reader (Vienna, VA, USA). Cells were seeded in a 12-well plate and incubated for 48?h. EdU assay was performed using the BeyoClick? EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime Inst Biotech, China). Briefly, cells were incubated with EdU working solution for 1.5?h. Then, cells were fixed with 4% (v/v) paraformaldehyde for 20?min at room temperature. Next, cells were permeabilized with 0.3% (v/v) Triton X-100 in PBS for 15?min at room temperature after washing with 3% (m/v) BSA PBS solution. Then, the cells were incubated with Click Reaction Buffer for 30?min at room temperature in the dark. Hoechst 33342 was added to each well and incubated for 10?min in the dark at room temperature. Finally, cells were photographed by fluorescence microscope (Zeiss, Jena, Germany). Transwell Assay Cell invasion was detected using transwell chambers (8-m pore size; Millipore). In brief, 600 l of complete medium was added to the bottom chamber, and 4 104 cells suspended in 200 l culture media with 10 mM metformin and 25 M celecoxib alone or in combination were placed in the upper chamber. A cotton swab was used to softly remove cells on the top surface of the membrane after 24?h. The upper chamber Y-33075 dihydrochloride was washed twice with PBS and then fixed in 4% (v/v) paraformaldehyde and stained in 0.1% (m/v) crystal violet solution for 30?min. Cells adhering to the bottom surface of the membrane were counted in five randomly selected areas under a 100 microscope field. All data were normalized with a control chamber that contained cells with no treatment. Wound Scratch Assay.