Mechanism of telomerase inhibitor BIBR1532 combined with autophagy inhibitor CQ in suppressing survival of melanoma cells

doi:10.19401/j.cnki.1007-3639.2025.05.001

Abstract

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

CLC Number:

R739.5

GONG Weihua, CHEN Lan, ZHAO Kun, KE Zhui, XU Qing, GUO Xianling. Mechanism of telomerase inhibitor BIBR1532 combined with autophagy inhibitor CQ in suppressing survival of melanoma cells[J]. China Oncology, 2025, 35(5): 431-439.

Figures/Tables 7

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

References 34

[1]	AHMED B, QADIR M I, GHAFOOR S. Malignant melanoma: skin cancer-diagnosis, prevention, and treatment[J]. Crit Rev Eukaryot Gene Expr, 2020, 30(4): 291-297.
[2]	RANDIC T, KOZAR I, MARGUE C, et al. NRAS mutant melanoma: towards better therapies[J]. Cancer Treat Rev, 2021, 99: 102238.
[3]	YANG F, XIAN R R, LI Y Y, et al. Telomerase reverse transcriptase expression elevated by avian leukosis virus integration in B cell lymphomas[J]. Proc Natl Acad Sci USA, 2007, 104(48): 18952-18957. doi: 10.1073/pnas.0709173104 pmid: 18024587
[4]	SONG Y J, ZHANG S S, GUO X L, et al. Autophagy contributes to the survival of CD133⁺ liver cancer stem cells in the hypoxic and nutrient-deprived tumor microenvironment[J]. Cancer Lett, 2013, 339(1): 70-81.
[5]	VISHWAKARMA K, DEY R, BHATT H. Telomerase: a prominent oncological target for development of chemotherapeutic agents[J]. Eur J Med Chem, 2023, 249: 115121.
[6]	JUDASZ E, LISIAK N, KOPCZYŃSKI P, et al. The role of telomerase in breast cancer’s response to therapy[J]. Int J Mol Sci, 2022, 23(21): 12844.
[7]	LI X H, HE S K, MA B Y. Autophagy and autophagy-related proteins in cancer[J]. Mol Cancer, 2020, 19(1): 12.
[8]	DEBNATH J, GAMMOH N, RYAN K M. Autophagy and autophagy-related pathways in cancer[J]. Nat Rev Mol Cell Biol, 2023, 24(8): 560-575.
[9]	KENIFIC C M, DEBNATH J. Cellular and metabolic functions for autophagy in cancer cells[J]. Trends Cell Biol, 2015, 25(1): 37-45. doi: 10.1016/j.tcb.2014.09.001 pmid: 25278333
[10]	LIU H, HE Z Y, VON RÜTTE T, et al. Down-regulation of autophagy-related protein 5 (ATG5) contributes to the pathogenesis of early-stage cutaneous melanoma[J]. Sci Transl Med, 2013, 5(202): 202ra123.
[11]	MIRACCO C, CEVENINI G, FRANCHI A, et al. Beclin 1 and LC3 autophagic gene expression in cutaneous melanocytic lesions[J]. Hum Pathol, 2010, 41(4): 503-512.
[12]	LAZOVA R, KLUMP V, PAWELEK J. Autophagy in cutaneous malignant melanoma[J]. J Cutan Pathol, 2010, 37(2): 256-268. doi: 10.1111/j.1600-0560.2009.01359.x pmid: 19615007
[13]	MAES H, MARTIN S, VERFAILLIE T, et al. Dynamic interplay between autophagic flux and Akt during melanoma progression in vitro[J]. Exp Dermatol, 2014, 23(2): 101-106.
[14]	LAZOVA R, CAMP R L, KLUMP V, et al. Punctate LC3B expression is a common feature of solid tumors and associated with proliferation, metastasis, and poor outcome[J]. Clin Cancer Res, 2012, 18(2): 370-379. doi: 10.1158/1078-0432.CCR-11-1282 pmid: 22080440
[15]	MAES H, AGOSTINIS P. Autophagy and mitophagy interplay in melanoma progression[J]. Mitochondrion, 2014, 19 Pt A: 58-68.
[16]	ASHRAFIZADEH M, MOHAMMADINEJAD R, TAVAKOL S, et al. Autophagy, anoikis, ferroptosis, necroptosis, and endoplasmic reticulum stress: potential applications in melanoma therapy[J]. J Cell Physiol, 2019, 234(11): 19471-19479. doi: 10.1002/jcp.28740 pmid: 31032940
[17]	PANGILINAN C, KLIONSKY D J, LIANG C Y. Emerging dimensions of autophagy in melanoma[J]. Autophagy, 2024, 20(8): 1700-1711.
[18]	CHUN-ON P, HINCHIE A M, BEALE H C, et al. TPP1 promoter mutations cooperate with TERT promoter mutations to lengthen telomeres in melanoma[J]. Science, 2022, 378(6620): 664-668.
[19]	TANIDA I, UENO T, KOMINAMI E. LC3 and autophagy[J]. Methods Mol Biol, 2008, 445: 77-88. doi: 10.1007/978-1-59745-157-4_4 pmid: 18425443
[20]	KURUSU R, FUJIMOTO Y, MORISHITA H, et al. Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex[J]. Dev Cell, 2023, 58(13): 1189-1205.e11. doi: 10.1016/j.devcel.2023.04.015 pmid: 37192622
[21]	BJØRKØY G, LAMARK T, PANKIV S, et al. Monitoring autophagic degradation of p62/SQSTM1[J]. Methods Enzymol, 2009, 452: 181-197. doi: 10.1016/S0076-6879(08)03612-4 pmid: 19200883
[22]	SHEN Z Y, WANG Y H, WANG G Z, et al. Research progress of small-molecule drugs in targeting telomerase in human cancer and aging[J]. Chem Biol Interact, 2023, 382: 110631.
[23]	ZVEREVA M I, SHCHERBAKOVA D M, DONTSOVA O A. Telomerase: structure, functions, and activity regulation[J]. Biochemistry (Mosc), 2010, 75(13): 1563-1583. pmid: 21417995
[24]	BERNARDES DE JESUS B, BLASCO M A. Telomerase at the intersection of cancer and aging[J]. Trends Genet, 2013, 29(9): 513-520. doi: 10.1016/j.tig.2013.06.007 pmid: 23876621
[25]	BERGER M F, HODIS E, HEFFERNAN T P, et al. Melanoma genome sequencing reveals frequent PREX2 mutations[J]. Nature, 2012, 485(7399): 502-506.
[26]	HUANG F W, HODIS E, XU M J, et al. Highly recurrent TERT promoter mutations in human melanoma[J]. Science, 2013, 339(6122): 957-959.
[27]	CHIBA K, LORBEER F K, HUNTER SHAIN A, et al. Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism[J]. Science, 2017, 357(6358): 1416-1420.
[28]	GUTERRES A N, VILLANUEVA J. Targeting telomerase for cancer therapy[J]. Oncogene, 2020, 39(36): 5811-5824. doi: 10.1038/s41388-020-01405-w pmid: 32733068
[29]	TARAZÓN E, DE UNAMUNO BUSTOS B, MURRIA ESTAL R, et al. miR-138-5p suppresses cell growth and migration in melanoma by targeting telomerase reverse transcriptase[J]. Genes (Basel), 2021, 12(12): 1931.
[30]	HRDLIČKOVÁ R, NEHYBA J, BARGMANN W, et al. Multiple tumor suppressor microRNAs regulate telomerase and TCF7, an important transcriptional regulator of the Wnt pathway[J]. PLoS One, 2014, 9(2): e86990.
[31]	CHO W C S. OncomiRs: the discovery and progress of microRNAs in cancers[J]. Mol Cancer, 2007, 6: 60. pmid: 17894887
[32]	WANG H, NI J, GUO X H, et al. Effects of folate on telomere length and chromosome stability of human fibroblasts and melanoma cells in vitro: a comparison of folic acid and 5-methyltetrahydrofolate[J]. Mutagenesis, 2023, 38(3): 160-168.
[33]	DAMM K, HEMMANN U, GARIN-CHESA P, et al. A highly selective telomerase inhibitor limiting human cancer cell proliferation[J]. EMBO J, 2001, 20(24): 6958-6968. pmid: 11742973
[34]	NOWOSAD A, JEANNOT P, CALLOT C, et al. p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth[J]. Nat Cell Biol, 2020, 22(9): 1076-1090. doi: 10.1038/s41556-020-0554-4 pmid: 32807902