DCPIB通过上调DDIT3抑制黑色素瘤细胞增殖的机制研究

doi:10.11904/j.issn.1002-3070.2025.02.005

实用肿瘤学杂志 ›› 2025, Vol. 39 ›› Issue (2): 99-107.doi: 10.11904/j.issn.1002-3070.2025.02.005

DCPIB通过上调DDIT3抑制黑色素瘤细胞增殖的机制研究

栗梦雅^1,2, 蔡梦迪^1,2

1.中国遗传资源保护与疾病防控教育部重点实验室(哈尔滨 150081);
2.哈尔滨医科大学医学遗传学研究室

收稿日期:2024-07-02 修回日期:2025-02-28 出版日期:2025-04-28 发布日期:2025-05-06
通讯作者: 蔡梦迪,E-mail:caimengdi@ems.hrbmu.edu.cn
作者简介:栗梦雅,女,(1998—),硕士研究生,从事DCPIB治疗黑色素瘤的机制研究。
基金资助:
中国遗传资源保护与疾病防控教育部重点实验室开放课题(编号:LPHGRDC2020-002);哈尔滨医科大学少帅揭榜项目(编号:HMUMIF-21007)

DCPIB inhibits melanoma cell proliferation by upregulating DDIT3

LI Mengya^1,2, CAI Mengdi^1,2

1. Key Laboratory of Preservation of Human Genetic Resources and Disease Control in China,Ministry of Education,Harbin 150081, China;
2. Laboratory of Genetics,Harbin Medical University

Received:2024-07-02 Revised:2025-02-28 Online:2025-04-28 Published:2025-05-06

摘要/Abstract

摘要： 目的探讨选择性可逆体积调节性阴离子通道抑制剂DCPIB(4-[(2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-3H-inden-5-yl)oxy]butanoic acid)对黑色素瘤细胞增殖能力的影响及作用机制。方法采用CCK-8法和EdU实验检测DCPIB对黑色素瘤细胞A375和RPMI-7951增殖能力的影响;通过转录组测序分析对照组(无水乙醇处理的A375细胞)和DCPIB处理组(10 μM DCPIB处理的A375细胞)的差异表达基因,寻找显著变化的信号通路;qRT-PCR检测差异表达基因mRNA水平变化;Western blot检测相关信号通路蛋白水平变化。结果 DCPIB可显著抑制人黑色素瘤细胞A375和RPMI-7951的增殖(P＜0.01)。转录组测序结果显示DCPIB处理组中DNA损伤诱导转录因子3(DNA damage-inducible transcript 3,DDIT3)显著上调(t=161.800,P＜0.001),多种DNA损伤修复途径相关关键基因Ku70、Ku80、RAD51、MLH1、MSH6、PARP1、FANCD2 mRNA表达水平下调,且qRT-PCR验证了DCPIB处理A375细胞36 h后上述DNA损伤修复通路关键基因的mRNA表达水平显著降低(P＜0.05)。GO功能富集和KEGG通路富集分析结果显示DCPIB处理后内质网应激相关基因HSPA5、DDIT3、ATF4、XBP1、ERN1、EIF2AK3 mRNA表达水平上调,下游PI3K-Akt信号通路表达异常,qRT-PCR验证DCPIB处理组A375细胞的DDIT3下游分子TRIB3表达上调(t=2.819,P=0.048),下游Akt-mTOR信号通路受到抑制,p-Akt(t=7.638,P=0.002)和p-mTOR(t=4.898,P=0.008)蛋白表达水平明显下降。结论 DCPIB可显著抑制黑色素瘤细胞的增殖,其机制可能与DDIT3-TRIB3-Akt-mTOR信号轴有关。

关键词: 黑色素瘤, A375细胞, 内质网应激, DNA损伤修复, Akt-mTOR信号通路

Abstract: Objective The Objective of this study was to investigate the effect of DCPIB(4-[(2-butyl-6,7-dichloro-2-cyclopentyl-1-oxo-3H-inden-5-yl)oxy]butanoic acid),a selective reversible volume-regulated anion channel inhibitor,on the proliferation capacity of melanoma cells and its mechanism of action. Methods CCK-8 assay and EdU assay were used to detect the effect of DCPIB on the proliferation capacity of A375 cells and RPMI-7951 cells.Transcriptome sequencing was conducted to analyze the differentially expressed genes(DEGs)between the control group(A375 cells treated with absolute ethanol)and the DCPIB-treated group(A375 cells treated with 10 μM of DCPIB)to find the significantly changed signaling pathways.qRT-PCR was used to detect the changes in the mRNA levels of DEGs.Western blot was used to detect the changes in the expression of proteins related to relevant signaling pathways. Results DCPIB significantly inhibited the proliferation of A375 cells and RPMI-7951 cells(P＜0.01).The results of transcriptome sequencing revealed that DNA damage-inducible transcript 3(DDIT3)was significantly upregulated in the DCPIB-treated group(t=161.800,P＜0.001),and the levels of multiple key genes mRNA related to DNA damage repair pathway,including Ku70,Ku80,RAD51,MLH1,MSH6,PARP1,FANCD2 were downregulated.qRT-PCR verified that the levels of key genes mRNA in the above DNA damage repair pathways were significantly reduced after DCPIB treatment in A375 cells for 36 h(P＜0.05).GO functional enrichment and KEGG pathway analyses revealed that after DCPIB treatment,the mRNA levels of endoplasmic reticulum stress-related genes,including HSPA5,DDIT3,ATF4,XBP1,ERN1,EIF2AK3 were upregulated,and the downstream PI3K-Akt signaling pathway was abnormally expressed.qRT-PCR verified that the expression of TRIB3,a downstream molecule of DDIT3 was upregulated in A375 cells treated with DCPIB(t=2.819,P=0.048),and the downstream Akt-mTOR signaling pathway was inhibited,and the levels of p-Akt(t=7.638,P=0.002)and p-mTOR proteins(t=4.898,P=0.008)were significantly decreased. Conclusion DCPIB can significantly inhibit the proliferation of melanoma cells,and its mechanism may be related to the DDIT3-TRIB3-Akt-mTOR signaling axis.

Key words: Melanoma, A375 cells, Endoplasmic reticulum stress, DNA damage repair, Akt-mTOR signaling pathway

中图分类号:

R739.5

栗梦雅, 蔡梦迪. DCPIB通过上调DDIT3抑制黑色素瘤细胞增殖的机制研究[J]. 实用肿瘤学杂志, 2025, 39(2): 99-107.

LI Mengya, CAI Mengdi. DCPIB inhibits melanoma cell proliferation by upregulating DDIT3[J]. Journal of Practical Oncology, 2025, 39(2): 99-107.

参考文献

1 Ahmed S,Alam W,Alsharif KF,et al.Therapeutic potential of marine peptides in malignant melanoma[J].Environ Res,2023,227:115771.
2 de Almeida Gonçalves VDM,de Almeida Camargo Filho MF,Zaleski T,et al.Chemotherapy in focus:a meta-analysis confronts immunotherapy in the treatment of advanced melanoma[J].Crit Rev Oncol Hematol,2021,161:103304.
3 Takahashi H,Yamada T,Denton JS,et al.Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly[J].eLife,2023,12:e82431.
4 Liu T,Stauber T.The volume-regulated anion channel LRRC8/VRAC is dispensable for cell proliferation and migration[J].Int J Mol Sci,2019,20(11):2663.
5 Zhang Y,Zhang H,Feustel PJ,et al.DCPIB,a specific inhibitor of volume regulated anion channels(VRACs),reduces infarct size in MCAo and the release of glutamate in the ischemic cortical penumbra[J].Exp Neurol,2008,210(2):514-520.
6 Wong R,Chen W,Zhong X,et al.Swelling-induced chloride current in glioblastoma proliferation,migration,and invasion[J].J Cell Physiol,2018,233(1):363-370.
7 Yoshimoto S,Matsuda M,Kato K,et al.Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells[J].Eur J Pharmacol,2021,895:173881.
8 Decher N,Lang HJ,Nilius B,et al.DCPIB is a novel selective blocker of ICl,swell and prevents swelling-induced shortening of guinea-pig atrial action potential duration[J].Br J Pharmacol,2001,134(7):1467-1479.
9 Lemay S-E,Bonnet S,Potus F.Commentary on:Xbp1s-Ddit3,DNA damage and pulmonary hypertension[J].Clin Sci,2022,136(1):163-166.
10 Zhang S,Xie R,Zhong A,et al.Targeted therapeutic strategies for melanoma[J].Chin Med J,2023,136(24):2923-2930.
11 Castro-Pérez E,Singh M,Sadangi S,et al.Connecting the dots:Melanoma cell of origin,tumor cell plasticity,trans-differentiation,and drug resistance[J].Pigment Cell Melanoma Res,2023,36(5):330-347.
12 Rydén V,El-Naggar AI,Koliadi A,et al.The role of dacarbazine and temozolomide therapy after treatment with immune checkpoint inhibitors in malignant melanoma patients:a case series and meta-analysis[J].Pigment Cell Melanoma Res,2024,37(3):352-362.
13 Zhang H,Cui S,Jing Z,et al.LRRC8A is responsible for exosome biogenesis and volume regulation in colon cancer cells[J].Biochem J,2023,480(9):701-713.
14 Zhou Z,Yin H,Suye S,et al.Fance deficiency inhibits primordial germ cell proliferation associated with transcription–replication conflicts accumulate and DNA repair defects[J].J Ovarian Res,2023,16(1):160.
15 Suba Z.DNA damage responses in tumors are not proliferative stimuli,but rather they are DNA repair actions requiring supportive medical care[J].Cancers,2024,16(8):1573.
16 Zhao W,Li G,Zhang Q,et al.Cardiac glycoside neriifolin exerts anti-cancer activity in prostate cancer cells by attenuating DNA damage repair through endoplasmic reticulum stress[J].Biochem Pharmacol,2023,209:115453.
17 Hounye AH,Hu B,Wang Z,et al.Evaluation of drug sensitivity,immunological characteristics,and prognosis in melanoma patients using an endoplasmic reticulum stress-associated signature based on bioinformatics and pan-cancer analysis[J].J Mol Med(Berl),2023,101(10):1267-1287.
18 Chen X,Cubillos-Ruiz JR.Endoplasmic reticulum stress signals in the tumour and its microenvironment[J].Nat Rev Cancer,2021,21(2):71-88.
19 Ediriweera MK,Tennekoon KH,Samarakoon SR.Role of the PI3K/AKT/mTOR signaling pathway in ovarian cancer:biological and therapeutic significance[J].Semin Cancer Biol,2019,59:147-160.
20 Huang T,Xu T,Wang Y,et al.Cannabidiol inhibits human glioma by induction of lethal mitophagy through activating TRPV4[J].Autophagy,2021,17(11):3592-3606.
21 Hu X,Pan G,Luo J,et al.Kuwanon H inhibits melanoma growth through cytotoxic endoplasmic reticulum stress and impaired autophagy flux[J].J Agric Food Chem,2023,71(37):13768-13782.

[1]	张塨钰, 郭浩兵, 鲁莉莉, 宋宁扬, 朴松林. POM121在头颈部黏膜黑色素瘤中的表达及临床意义[J]. 实用肿瘤学杂志, 2025, 39(2): 108-115.
[2]	李勇, 刘冰梅. 构建基于染色质重塑相关基因的皮肤黑色素瘤预后预测模型[J]. 实用肿瘤学杂志, 2025, 39(2): 134-143.
[3]	李路桥, 邹征云. 恶性黑色素瘤免疫治疗超进展研究现状[J]. 实用肿瘤学杂志, 2023, 37(2): 144-148.
[4]	胡启辉, 杜毅超, 贺凯, 付文广, 夏先明. 内质网应激在肝癌中的作用[J]. 实用肿瘤学杂志, 2021, 35(2): 160-164.
[5]	王灿, 罗茜, 罗焱, 刘素桃, 余音, 刁庆春, 黎智, 李晶. 甲基化修饰抑癌基因MSX1抑制黑色素瘤细胞迁移和侵袭的研究[J]. 实用肿瘤学杂志, 2020, 34(6): 511-517.
[6]	秦岚群, 邹征云. 恶性黑色素瘤化疗研究进展[J]. 实用肿瘤学杂志, 2019, 33(2): 167-172.
[7]	刘雅菁, 刘国钰, 王树滨, 方征宇. 盐霉素对人黑色素瘤M21细胞增殖及自噬流的影响[J]. 实用肿瘤学杂志, 2018, 32(3): 203-207.
[8]	欧阳仲瑞, 柴双, 赵海杞, 刘耀华. 内质网应激PERK通路在肿瘤中的研究进展[J]. 实用肿瘤学杂志, 2018, 32(1): 38-41.
[9]	张嘉辉综述, 毕良佳审校. 光动力疗法治疗非黑色素瘤皮肤癌的研究进展[J]. 实用肿瘤学杂志, 2016, 30(1): 71-74.
[10]	吴红宇,祁文,徐振明,林文秀,杜娜,项阳. 幼儿先天性色素痣恶变并淋巴结转移性黑色素瘤1例[J]. 实用肿瘤学杂志, 2013, 27(5): 430-432.
[11]	胡萍萍,金春晖,倪依群,周留勇,王旺胜,黄箫娜. 右足底恶性黑色素瘤术后十二指肠转移内镜像个案报道[J]. 实用肿瘤学杂志, 2013, 27(2): 165-166.
[12]	冯和林, 郑丽华, 单保恩. 瘦素及其受体与黑色素瘤的研究进展[J]. 实用肿瘤学杂志, 2012, 26(6): 556-558.
[13]	姬巧霞, 汪治宇, 王玉华. 恶性黑色素瘤的凋亡相关基因^△[J]. 实用肿瘤学杂志, 2011, 25(1): 76-78.
[14]	成秉辰. 千里光总碱和千里光碱对体外培养的小鼠黑色素瘤细胞增殖的影响[J]. 实用肿瘤学杂志, 2009, 23(1): 45-47.
[15]	刘红胜, 章美珍, 徐锦屏, 金利刚. 10例肛管直肠恶性黑色素瘤临床病理免疫组化研究[J]. 实用肿瘤学杂志, 2008, 22(6): 506-508.

DCPIB通过上调DDIT3抑制黑色素瘤细胞增殖的机制研究

DCPIB inhibits melanoma cell proliferation by upregulating DDIT3

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价