NSAIDs inhibit autophagy flux and sensitize anti-tumor agent induced cell death to cancer cell lines

Abstract

Autophagy is a process in which double-membraned autophagosomes digest unnecessary organelles or proteins in cells and decompose them through lysosomes. If they do not function normally, the homeostasis of the cells is broken and causes various diseases. In addition, the activity of autophagy was significantly increased in the stress conditions such as hypoxia, chemotherapy, and irradiation in cancer cells, and the cancer cells had resistance to the anticancer drugs through the increased autophagy activity. Therefore, when a drug that inhibits autophagy is administered in combination with an anticancer drug, an increased effect can be expected. Previous studies have shown that diclofenac, one of the NSAIDs, inhibits autophagy. Therefore, the effect of the other 20 NSAIDs on the autophagy was confirmed by Western blotting with the half-value of IC50 in mouse normal hepatocyte AML12, and the autophagy markers LC3 and p62 were screened. Cytotoxicity of combination of 5 NSAIDs (diclofenac, aceclofenac, naproxen, dexibuprofen, flurbiprofen) with sorafenib, tamoxifen, 5-FU, and paclitaxel, in liver cancer cell lines HepG2 and Huh7, breast cancer cell line mcf7, colon cancer cell line HCT116 and lung cancer cell line A549 were observed through HCS instrument cyation. In addition, the combination of diclofenac and sorafenib in Huh7 was associated with changes in apoptosis through annexin V-PI assay, cleaved caspase 3, cleaved PARP and Bcl-2. Most of the NSAIDs showed autophagy inhibitory activity as a result of screening in AML12. The combination of sorafenib or tamoxifen with diclofenac, aceclofenac, naproxen, dexibuprofen, and flurbiprofen in HepG2 and mcf7, combination of sorafenib and diclofenac in Huh7, diclofenac, aceclofenac, dexibuprofen and 5-FU in HCT116, a combination of dexibuprofen, naproxen and paclitaxel in A549, showed a large synergistic effect of cell death. In addition, the cotreatment of diclofenac and sorafenib in Huh7 significantly increased apoptosis by increased apoptosis positive cells and increased apoptosis marker cleaved caspase 3, cleaved PARP also decrease of Bcl-2. As a result, diclofenac inhibited autophagy in hepatocellular carcinoma cells with increased autophagy activity through anticancer drugs, and it was confirmed that this increased cancer cell death. In addition, other NSAIDs show autophagy inhibiting activity, and it is presumed that increasing cell death when combined with an anticancer agent is due to autophagy inhibition. Therefore, it is expected that the combined use of NSAIDs and anticancer drugs will increase the susceptibility of existing chemotherapeutic agents to cancer treatment.