端粒酶抑制剂BIBR1532联合自噬抑制剂CQ抑制黑色素瘤细胞生存的机制研究

龚卫华; 陈兰; 赵昆; 柯追; 许青; 郭献灵

doi:10.19401/j.cnki.1007-3639.2025.05.001

您当前的位置：

首页 >

文章列表页 >

端粒酶抑制剂BIBR1532联合自噬抑制剂CQ抑制黑色素瘤细胞生存的机制研究

论著 | 更新时间：2025-12-31

- 端粒酶抑制剂BIBR1532联合自噬抑制剂CQ抑制黑色素瘤细胞生存的机制研究
- Mechanism of telomerase inhibitor BIBR1532 combined with autophagy inhibitor CQ in suppressing survival of melanoma cells
- 中国癌症杂志 2025年35卷第5期页码：431-439
- 作者机构：
  
  1. 上海市第十人民医院崇明分院肿瘤科，上海 202157
  2. 同济大学附属第十人民医院肿瘤科，上海 200072
  3. 安徽理工大学医学院，安徽淮南 232063
- 作者简介：
  
  [ "龚卫华（ORCID: 0009-0004-3312-3653），学士，主治医师，上海市第十人民医院崇明分院肿瘤科常务主任、综合接待办主任。" ]
  郭献灵（ORCID: 0000-0002-4785-2994 ），博士，副主任医师。
- 基金信息：
  
  崇明区科学技术委员会科技项目(CKY2018-34)
- DOI：10.19401/j.cnki.1007-3639.2025.05.001
  中图分类号： R739.5
- 收稿：2024-10-15，
  
  修回：2024-12-23，
  
  纸质出版：2025-05-30
- 稿件说明：
移动端阅览
龚卫华, 陈兰, 赵昆, 等. 端粒酶抑制剂BIBR1532联合自噬抑制剂CQ抑制黑色素瘤细胞生存的机制研究[J]. 中国癌症杂志, 2025,35(5):431-439.

Weihua GONG, Lan CHEN, Kun ZHAO, et al. Mechanism of telomerase inhibitor BIBR1532 combined with autophagy inhibitor CQ in suppressing survival of melanoma cells[J]. China Oncology, 2025, 35(5): 431-439.
龚卫华, 陈兰, 赵昆, 等. 端粒酶抑制剂BIBR1532联合自噬抑制剂CQ抑制黑色素瘤细胞生存的机制研究[J]. 中国癌症杂志, 2025,35(5):431-439. DOI： 10.19401/j.cnki.1007-3639.2025.05.001.

Weihua GONG, Lan CHEN, Kun ZHAO, et al. Mechanism of telomerase inhibitor BIBR1532 combined with autophagy inhibitor CQ in suppressing survival of melanoma cells[J]. China Oncology, 2025, 35(5): 431-439. DOI： 10.19401/j.cnki.1007-3639.2025.05.001.

摘要

背景与目的：

黑色素瘤是一种源于黑色素细胞的高度侵袭性恶性肿瘤，严重威胁人类的生命健康，其发病率和死亡率近年来持续升高。而端粒酶和自噬在细胞增殖、生存及应激反应中发挥关键作用。端粒酶通过延长染色体末端的端粒来维持细胞的复制能力；自噬作为一种细胞自我降解机制，既能帮助细胞清除受损成分以促进存活，也能在特定条件下诱导细胞死亡。在肿瘤微环境中，端粒酶和自噬常被异常激活或调控失衡，参与包括黑色素瘤在内的多种恶性肿瘤的发生、发展。本研究初步探究端粒酶和自噬在黑色素瘤发展中的作用，并评估端粒酶抑制剂BIBR1532联合自噬抑制剂氯喹（chloroquine，CQ）在治疗黑色素瘤中的潜在协同作用。

方法：

通过给予恶性黑色素瘤细胞A375端粒酶抑制剂BIBR1532处理，利用细胞计数试剂盒-8（cell counting kit-8，CCK-8）实验来评估细胞活力，并采用Annexin Ⅴ/碘化丙啶（propidium iodide，PI）双染色来检测细胞凋亡情况。此外，通过蛋白质印迹法（Western blot）检测自噬相关蛋白LC3-Ⅱ和p62的表达，利用自噬双标腺病毒转染技术来观察自噬流的变化。在此基础上，进一步联合应用BIBR1532和自噬抑制剂CQ，分析细胞增殖、凋亡率、线粒体膜电位变化和细胞周期分布，并通过克隆形成实验来验证细胞增殖能力，从而全面地评估这种联合治疗策略的疗效。

结果：

端粒酶抑制剂BIBR1532在50 μmol/L的浓度下能够显著抑制恶性黑色素瘤细胞A375的生长，并诱导细胞发生凋亡。同样在这一浓度下，BIBR1532能够上调A375细胞中自噬相关蛋白LC3-Ⅱ的表达，同时降低p62蛋白的表达。通过自噬双标腺病毒转染A375细胞，观察到经BIBR1532处理后，自噬流显著增强。BIBR1532（50 μmol/L）与自噬抑制剂CQ（20 μmol/L）的联合应用可显著促进A375细胞的死亡，诱导细胞凋亡和线粒体膜电位的破坏，导致细胞周期在G

/M期停滞，并显著抑制细胞的克隆形成能力。

结论：

端粒酶抑制剂BIBR1532不仅能抑制恶性黑色素瘤细胞增殖，还能激活这些细胞的自噬过程，而通过应用自噬抑制剂CQ抑制自噬反应则能增强恶性黑色素瘤细胞对端粒酶抑制剂BIBR1532的敏感性。

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 mor

bidity 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.

关键词

Keywords

references

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 .

RANDIC T , KOZAR I , MARGUE C , et al. NRAS mutant melanoma: towards better therapies [J ] . Cancer Treat Rev , 2021 , 99 : 102238 .

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 http://doi.org/10.1073/pnas.0709173104

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 .

VISHWAKARMA K , DEY R , BHATT H . Telomerase: a prominent oncological target for development of chemotherapeutic agents [J ] . Eur J Med Chem , 2023 , 249 : 115121 .

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.

LI X H , HE S K , MA B Y . Autophagy and autophagy-related proteins in cancer [J ] . Mol Cancer , 2020 , 19 ( 1 ): 12.

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 .

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 http://doi.org/10.1016/j.tcb.2014.09.001

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.

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 .

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 http://doi.org/10.1111/j.1600-0560.2009.01359.x

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 .

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 http://doi.org/10.1158/1078-0432.CCR-11-1282

MAES H , AGOSTINIS P . Autophagy and mitophagy interplay in melanoma progression [J ] . Mitochondrion , 2014 , 19 Pt A: 58 - 68 .

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 http://doi.org/10.1002/jcp.28740

PANGILINAN C , KLIONSKY D J , LIANG C Y . Emerging dimensions of autophagy in melanoma [J ] . Autophagy , 2024 , 20 ( 8 ): 1700 - 1711 .

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 .

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 http://doi.org/10.1007/978-1-59745-157-4_4

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 http://doi.org/10.1016/j.devcel.2023.04.015

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 http://doi.org/10.1016/S0076-6879(08)03612-4

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 .

ZVEREVA M I , SHCHERBAKOVA D M , DONTSOVA O A . Telomerase: structure, functions, and activity regulation [J ] . Biochemistry (Mosc) , 2010 , 75 ( 13 ): 1563 - 1583 .

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 http://doi.org/10.1016/j.tig.2013.06.007

BERGER M F , HODIS E , HEFFERNAN T P , et al. Melanoma genome sequencing reveals frequent PREX2 mutations [J ] . Nature , 2012 , 485 ( 7399 ): 502 - 506 .

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 .

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 .

GUTERRES A N , VILLANUEVA J . Targeting telomerase for cancer therapy [J ] . Oncogene , 2020 , 39 ( 36 ): 5811 - 5824 . DOI: 10.1038/s41388-020-01405-w http://doi.org/10.1038/s41388-020-01405-w

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.

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.

CHO W C S . OncomiRs: the discovery and progress of microRNAs in cancers [J ] . Mol Cancer , 2007 , 6 : 60 .

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 .

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 .

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 http://doi.org/10.1038/s41556-020-0554-4

浏览量

1107

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

uPAR通过MAPK信号抑制细胞自噬促进胰腺癌增殖、侵袭及化疗抵抗的作用研究

HMGA2对软脑膜转移黑色素瘤细胞迁移和增殖的影响

NRAS突变型晚期黑色素瘤的治疗进展

乳腺癌中心体调控蛋白SEC23B在肿瘤浸润转移中的作用及其机制研究

干预自噬影响DPD的表达促进5-FU抗结直肠癌疗效的实验研究