关键词: 黑色素瘤, 端粒酶, 端粒酶抑制剂, BIBR1532, 自噬, 自噬抑制剂, 氯喹, 肿瘤治疗

Abstract:

Background and purpose: Melanoma is a highly invasive malignant tumor originating from melanocytes, which poses a great threat to human life and health around the world, and its morbidity and mortality have been rising continuously in recent years. Telomerase and autophagy play crucial roles in cell proliferation, survival and stress response. Telomerase maintains the replication ability of cells by prolonging telomeres at the ends of chromosomes, and autophagy, as a self-degradation mechanism of cells, can not only help cells remove damaged components to promote survival, but also induce cell death under certain conditions. In the tumor environment, they are often abnormally activated or out of balance, and participate in the occurrence and development of many cancers, including melanoma. This study investigated the roles of telomerase and autophagy in melanoma progression and evaluated the potential synergistic therapeutic effects of combined application of telomerase inhibitor BIBR1532 and autophagy inhibitor chloroquine (CQ) in melanoma treatment. Methods: Malignant melanoma cells A375 were treated with telomerase inhibitor BIBR1532. The cell viability was assessed using the cell counting kit-8 (CCK-8) assay, and the cell apoptosis was detected using the Annexin Ⅴ/propidium iodide (PI) double staining method. Additionally, the expressions of autophagy-related proteins LC3-Ⅱ and p62 were detected by Western blot, and the changes in autophagy flux were observed using dual-tagged adenovirus transfection technology. Based on these studies, BIBR1532 and the autophagy inhibitor CQ were further applied in combination to analyze cell proliferation, apoptotic rate, changes in mitochondrial membrane potential, and cell cycle distribution, and the cloning formation experiment was used to verify the cell's proliferative capacity, thereby comprehensively evaluating the efficacy of this combined treatment strategy. Results: Telomerase inhibitor BIBR1532 at a concentration of 50 μmol/L significantly inhibited the growth of malignant melanoma cells A375 and induced apoptosis. At the same concentration, BIBR1532 upregulated the expression of the autophagy-related protein LC3-Ⅱ in A375 cells, while downregulating the expression of p62 protein. By transducing A375 cells with a dual-tagged adenovirus, it was observed that autophagy flux was significantly enhanced after treatment with BIBR1532. Furthermore, the combined application of BIBR1532 (50 μmol/L) and the autophagy inhibitor CQ (20 μmol/L) significantly promoted the death of A375 cells, induced apoptosis and destruction of mitochondrial membrane potential, caused cell cycle arrest at the G2/M phase, and significantly inhibited the cell’s clonogenic ability. Conclusion: Telomerase inhibitor BIBR1532 not only inhibits the proliferation of malignant melanoma cells but also activates the autophagy process in these cells, and inhibition of the autophagy response by autophagy inhibitor CQ can enhance the sensitivity of malignant melanoma cells to telomerase inhibitor BIBR1532.

Key words: Melanoma, Telomerase, Telomerase inhibitor, BIBR1532, Autophagy, Autophagy inhibitor, Chloroquine, Tumor therapy