Study On The Mechanism Of The Increased Cytotoxicity Of Cisplatin By Baf A1 In Tongue Squamous Cell Carcinoma Cells

Objective: Cisplatin is the basic drug for the chemotherapy of tongue squamous cell carcinoma(TSCC), but cisplatin resistance is clinically common. Recent studies found that cisplatin cytotoxicity decreased in ovarian cancer, lung cancer, glioma and other tumors was due to autophagy, while whether autophagy affect the sensitivity of cisplatin in TSCC was elusive. The present study was to investigate the relationship between autophagy and cisplatin sensitivity in TSCC, and to further study the underlying mechanism.Methods: Western blot was used to detect the basal autophagy level in TSCC cell lines, the expression of DNA damage marker p-H2A.X and the related signal pathway protein after cisplatin treated. MTT assay was used to test the cytotoxicity of cisplatin and cisplatin combined with autophagy inhibitors on TSCC cells. The apoptosis-inducing effect and the intracellular reactive oxygen species (ROS) level in TSCC cells after different treatment of cisplatin and autophagy inhibitors were detected by flow cytometry. The expression of lysosomal marker in cis-resistant cell lines was detected by immunofluorescence and confocal microscopy. Real-time PCR was performed for examming the mRNA level of transcription factor EB (TFEB) in cis-resistant cells and non-resistant cells treated by cisplatin. The complete lysosomes in TSCC cells were extracted by the lysosome isolation kit. ICP-MS was employed to analyze platinum ions content which were binded with DNA and uptaken by lysosomes. The distribution of TFEB in the nucleus and cytoplasm was detected by subcellular fractionation assay.Results: The basal autophagy level of TSCC cells was significantly higher than that of normal tongue epithelium cells. Bafilomycin Al(Baf A1) and Chloroquine(CQ)pretreatment could increase the sensitivity of TSCC cells to cisplatin, but knock-out of Atg5 or combined with other autophagy inhibitor could not get similar effect. Baf A1 combined with cisplatin significantly increased DNA binding platinum ions, DNA damage and intracellular ROS level. The lysosome number in the cis-resistant cells and the cis-treated cells was increased compared with the control group.Cisplatin-induced TFEB nuclear translocation promoted lysosomal biosynthesis and was regulated by c-Abl, but not by mTORC1 or ERK2. Silence of TFEB expression elevated the sensitivity of TSCC cells to cisplatin.Conclusion: In TSCC cells, lysosomal biosynthesis is enhanced through c-Abl/TFEB parthway after cisplatin treated, leading to more lysosomal uptake of platinum ions and reduction of platinum ions bingding to DNA, thereby reduces the cytotoxic effect of cisplatin. Whereas Baf A1 improves the cytotoxicity of cisplatin in TSCC cells by inhibiting the capcivity of lysosome uptake and increasing DNA damage and intracellular ROS level. Howerver , this synergistic effect is independent of autophagy.