The Role Of Autophagic Flux In Cisplatin Resistance Of Human Ovarian Carcinoma Cells Through ATP-mediated Lysosomal Function

In the treatment of ovarian cancer, platinum-based cisplatin is a first-line therapeutic agent, and achieves some therapeutic effect. With the widespread use of cisplatin in ovarian cancer, ovarian cancer cells gradually acquires cisplatin resistance, which is the difficulty of the clinical treatment. Cisplatin induces DNA damage to trigger cell cycle arrest and initiate cell apoptosis. Several recent studies demonstrate endoplasmic reticulum(ER) stress and autophagy can promote cisplatin resistance in cancer cells.Autophagy is characterized by sequestration of cytoplasmic components in autophagosomes. These autophagosomes subsequently fuse with lysosomes to form autolysosomes, in which cytoplasmic materials including organelles are degraded. Under many physiological and pathological conditions, autophagy is induced, and it often promotes survival of cancer cells. Studies have shown that excessive or sustained autophagy results in the death of cancer cell. Autophagy may play dual roles in tumor cells, but the exact mechanism of autophagy remains controversial. Autophagy is a lysosomal-dependent process, and autophagosomes need to fuse with lysosomes at the end of autophagy, which ensures completion of autophagy flux. In the autophagy process, the fusion of autophagosome with lysosome consumes large amounts of lysosomes. Further research finds that autophagy may be involved in chemotherapeutics resistance including cisplatin. However, the number and function of lysosomesis is required for maintaining autophagic flux. Thus we hypothesize that autophagy- lysosome may be associated with chemotherapeutics resistance of cancer cells.Studies show that lysosomes contain rich ATP, and lysosome is also a cellular degradative center. Lysosomal lower p H environment and hydrolytic enzymes are critical to the function of lysosomal degradation, but ATP provides energy for lysosomal acidic environment and lysosomal protease activation. The acidic environment within the lysosome is a necessary condition for lysosomal protease activation, however, V-ATP ase is required for lysosomal acidic environment, and the function of V-ATPase protein complex need ATP. High expression of part lysosomal hydrolases also is associated with cancer, especially malignant tumors often highly express Cathepsin D. Cathepsin D plays an important role in the cell, and its deficiency triggers cell death, and may also lead to nerve lesions. And the activity of Cathepsin D in lysosomes needs ATP to provide energy. Therefore, we speculate that reducing lysosomal ATP may result in lysosomal dysfunction, lower the function of autolysosome, affect autophagy flux, and even lead to lysosomal hydrolases released within the cytoplasm to trigger cell apoptosis. Thus, lysosome may be an important determinant in the regulation of tumor chemoresistance through ATP mediated-lysosomal function.In this study, we chose human ovarian cancer SKOV3 cells(parental) and SKOV3/DDP cells(cisplatin resistance) as objects of study; based on ATP mediated-lysosome exploring the mechanism of autophagic flux in cisplatin resistance of ovarian cancer, and providing new strategies for cancer treatment. Method:(1) MTT assay detects cell viability of human ovarian cancer SKOV3 and SKOV3/DDP cells treated with cisplatin.(2) After cisplatin treatment in human ovarian carcinoma SKOV3 and SKOV3 cells, western blot detects the expression of Cleaved Caspase-3, p62, LC3, LAMP1, Cathepsin D; indirect immunofluorescence observes the colocalization of p62 and LC3 or Ub.(3) Confocal laser microscopy observed that the alteration of LTR staining in SKOV3 and SKOV3/DDP cells, and the colocalization of LAMP1 and LC3.(4) Cathepsin D activity of SKOV3 and SKOV3/DDP cells in cell lysates is detected using a fluorometric cathepsin D activity assay kit.(5) After inhibiting autophagy by 3-MA or CQ, MTT assay detects cell viability of SKOV3/DDP cells; western blot analysis the expression of p62, LC3, Ub, Bcl-2, Bax, Cleaved Caspase-3, Cathepsin D and t Bid; confocal laser microscopy observes the colocalization of LAMP1 and LC3, and the alteration of LTR staining; assay kit detects Cathepsin D activity; flow cytometry evaluates the rate of apoptosis cells.(6) After inhibiting Cathepsin D activity by Pep A, western blot detects the expression of p62, LC3 and Ub; assay kit detects the alteration of Cathepsin D activity; indirect immunofluorescence detects the colocalization of LAMP1 and LC3.(7) Confocal laser microscopy detects lysosomal ATP within cells by Quinacrine staining. After inhibiting the accumulation of lysosomal ATP by DIDS, confocal laser microscopy observes LTR staining; western blot detects the expression of p62 and LC3; indirect immunofluorescence detects the colocalization of LAMP1 and LC3. Results 1. Compared with SKOV3 cells, SKOV3/DDP cells were relatively insensitive to cisplatin; cisplatin could induce the expression of LC3-II protein in SKOV3 and SKOV3/DDP cells; compared with SKOV3 cells, cisplatin induced higher expression of LC3-II protein in SKOV3/DDP cells at 12h; meanwhile, indirect immunofluorescence also observed the colocalization of p62 and LC3. 2. Compared with SKOV3 cells, the expression of lysosomal membrane associated protein LAMP1 and Cathepsin D was higher; cisplatin enhanced LTR staining and Cathepsin D activity. 3. Compared with the autophagy inhibitor 3-MA combination with cisplatin, targeting lysosomal autophagy inhibitor CQ combination with cisplatin significantly decreased the rate of cell viability, enhanced the expression of p62, LC3-II and ubiquitinated proteins, reduced LTR staining and Cathepsin D activity, or even triggered the mitochondria-mediated apoptosis. 4. In SKOV3/DDP cells, Cathepsin D active inhibitor Pep A combination with cisplatin significantly reduced Cathepsin D activity, weakened LTR staining, but also raised the expression of p62, LC3-II and ubiquitinated proteins. 5. Compared with SKOV3 cells, SKOV3/DDP cells contained richer ATP. After DIDS inhibiting accumulation of lysosomal ATP in SKOV3/DDP cells, it significantly reduced the content of lysosomal ATP, but also raised the expression of p62 and LC3-II protein, increased the colocalization of LAMP1 and LC3, reduced LTR staining and Cathepsin D activity. Conclusions: 1. Cisplatin can induce autophagy in human ovarian cancer SKOV3 and SKOV3/DDP cells. Compared with SKOV3 cells, the level of autophagy in SKOV3/DDP cells was higher, suggesting that autophagy may be associated with cisplatin resistance of ovarian cancer cells. Further research found in SKOV3/DDP cells treated with or without cisplatin, the expression of lysosomal membrane associated protein(LAMP1) and lysosomal critical Cathepsin D were high; cisplatin enhanced LTR staining and the Cathepsin D activity. These results showed compared SKOV3 cells, SKOV3/DDP cells contained abundant lysosomes and Cathepsin D, suggesting that autophagylysosomal pathway may be associated with cisplatin resistance of ovarian cancer cells. 2. Compared with the autophagy inhibitor 3-MA combination with cisplatin, targeting lysosomal autophagy inhibitor CQ combination with cisplatin significantly decreased the rate of cell viability, increased the expression of p62, LC3-II and ubiquitinated proteins, reduced LTR staining and Cathepsin D activity, caused the accumulation of autolysosomes, and even triggered the mitochondria-associated apoptosis, indicating lysosomal dysfunction inhibited autophagy flux, and increased SKOV3/DDP cells to cisplatin sensitivity, suggesting that autophagy protected ovarian cancer cells from cisplatin-induced apoptosis, and lysosomes may be involved in cisplatin resistance of ovarian cancer cells through regulating autophagy flux. 3. Compared with SKOV3 cells, SKOV3/DDP cells contained richer Cathepsin D. Inhibiting Cathepsin D activity increased the expression of p62 and LC3-II protein, reduced LTR staining and Cathepsin D activity. These results suggest Lysosomal Cathepsin D ensured autophagy flux through maintaining the function of lysosomal degradation, and promoted the role of autophagy flux in cisplatin resistance of ovarian cancer cells. 4. Compared to SKOV3 cells, the lysosomes of SKOV3/DDP cells contained richer ATP. After DIDS inhibiting lysosomal ATP accumulation of SKOV3/DDP cells, it significantly increased the expression of autophagy- associated proteins, reduced LTR staining and Cathepsin D activity, and even caused the accumulation of autolysosomes, suggesting that reducing lysosomal ATP not only affectd lysosomes, but also disturbed the fuction of autolysosomal degradation, and regulating the role of autophagy flux in cisplatin resistance of ovarian cancer cells.In this study, for the first time, based on ATP mediated-lysosome exploring the mechanism of autophagy flux in cisplatin resistance of ovarian cancer, and providing new strategies for overcoming chemotherapeutic resistance of ovarian cancer.