组蛋白去乙酰化酶抑制剂CUDC-101对前列腺癌DU145细胞DNA损伤、迁移和上皮-间质转化的影响

doi:10.13481/j.1671-587X.20240213

摘要/Abstract

摘要：

目的探讨泛素化组蛋白（H2AX）在前列腺癌（PCa）和癌旁良性前列腺组织中的表达及与其PCa患者临床病理参数之间的关系，阐明新型组蛋白去乙酰化酶抑制剂（HDACi）CUDC-101对PCa的DNA损伤、迁移及上皮-间质转化（EMT）的影响。方法肿瘤基因组谱（TCGA）数据库和UALCAN数据库检索各种癌症组织中H2AX mRNA的表达情况及其在PCa与正常前列腺组织中的表达差异，分析其表达与PCa患者临床预后的关系；采用免疫组织化学染色法检测在PCa组织和癌旁良性前列腺组织中H2AX蛋白的表达情况，分析H2AX蛋白表达与PCa患者临床病理参数的关系；体外培养DU145细胞，分为对照组、5% FBS组、5% FBS+100 μmol·L^-1CUDC-101组和5% FBS+200 μmol·L^-1CUDC-101组，采用细胞划痕实验和Transwell小室实验检测CUDC-101处理前后PCa DU145细胞的细胞划痕面积及迁移细胞数；免疫荧光染色法检测CUDC-101处理后PCa DU145细胞中上皮细胞标志物E钙黏蛋白（E-cadherin）和磷酸化组蛋白γ-H2AX表达情况；Western blotting法检测CUDC-101处理后EMT相关蛋白、γ-H2AX和磷酸化蛋白激酶B（p-AKT）表达情况。结果 TCGA数据库和UALCAN数据库分析，H2AX mRNA在PCa组织中高表达，并且H2AX低表达组患者的无病生存期（DFS）明显高于H2AX mRNA高表达组（P<0.001）；免疫组织化学染色，在PCa组织中H2AX蛋白强阳性表达率高于在癌旁良性前列腺组织（64.34% vs 14.29%），其过表达与PCa的T分期（P=0.001）和世界卫生组织/国际泌尿科病理学会（WHO/ISUP）预后分级分组（P=0.004）有关，但与PCa患者的年龄、Gleason评分、淋巴结转移、神经和脉管浸润无关（P>0.05）；免疫荧光染色法检测，与对照组和EMT诱导组比较，CUDC-101处理组E-cadherin和γ-H2AX蛋白的荧光表达增强；细胞划痕实验和Transwell小室实验检测，与对照组比较，CUDC-101处理组DU145细胞愈合面积和迁移细胞数明显下调；Western blotting法检测，与EMT诱导组比较，CUDC-101处理组E-cadherin和γ-H2AX蛋白表达水平升高P<0.05或P<0.01），波形蛋白（Vimentin）和p-AKT蛋白表达水平降低（P<0.05或P<0.01）。结论 H2AX蛋白过表达与PCa患者的不良预后密切关联。新型HDACi抑制剂CUDC-101可调控H2AX和AKT的磷酸化，抑制PCa细胞EMT过程。

关键词: CUDC-101, 泛素化组蛋白, 前列腺肿瘤, 上皮-间质转化, DNA损伤

Abstract:

Objective To discuss the expression of ubiquitinated histone （H2AX） in the prostate cancer （PCa） and adjacent benign prostate tissues， and its relationship with the clinicopathological parameters of the PCa patients，and to clarify the effect of the novel histone deacetylase inhibitor （HDACi） CUDC-101 on the DNA damage， migration， and epithelial-mesenchymal transition （EMT） in PCa. Methods The expression levels of H2AX mRNA in various cancer tissues were retrieved from The Cancer Genome Atlas （TCGA） and UALCAN Databases to analyze the expression differences between PCa and normal prostate tissues and its connection with the clinical prognosis of the patients with PCa； immunohistochemistry method was used to detect the expression of H2AX protein in PCa tissue and adjacent benign prostate tissue， and its relationship with the clinicopathological parameters of the PCa patients was analyzed. The DU145 cells were cultured in vitro and divided into control group， 5% FBS group， 5% FBS+100 μmol·L^-1 CUDC-101 group， and 5% FBS+200 μmol·L^-1 CUDC-101 group.Cell scratch assay and Transwell chamber assay were used to detect the scratch area of the cells and the number of migration cells in the PCa DU145 cells before and after treated with CUDC-101； immunofluorescence staining was used to detect the expressions of epithelial cell marker E-cadherin （E-cadherin） and phosphorylated histone γ-H2AX in the PCa DU145 cells after treated with CUDC-101； Western blotting method was used to detect the expression levels of EMT-related protein， γ-H2AX， and phosphorylated protein kinase B （p-AKT） in the PCa DU145 cells after treated with CUDC-101. Results The TCGA Database and UALCAN Database analysis results showed that H2AX mRNA was highly expressed in the PCa tissue， and the disease-free survival （DFS） of the patients with low expression of H2AX was longer than those patients with high expression of H2AX mRNA （P<0.001）； the immunohistochemistry results showed that compared with adjacent benign prostate tissue，the rate of strong positive expression of H2AX protein in PCa tissue was increased （64.34% vs 14.29%）， and its over-expression was associated with the T stage of PCa （P=0.001） and World Health Organization（WHO）/International Society of Urological Pathology（ISUP） prognostic grapde group（GG）（P=0.004）， but was not associated with the patients’ age， Gleason score， lymphnode metastasis， or nerve and vascular invasion （P>0.05）； the immunofluorescence staining results showed that the compared with control and EMT induction groups，the fluorescence expressions of E-cadherin and γ-H2AX proteins in CUDC-101 treatment group were increased； the cell scratch assay and Transwell chamber assay results showed that compared with control group， the scratch healing area and number of migration DU145 cells in CUDC-101 treatment group was significantly decreased； the Western blotting results showed that compared with EMT induction group， the expression levels of E-cadherin and γ-H2AX proteins in the cells in CUDC-101 treatment group were increased （P<0.05 or P<0.01）， while the expression levels of Vimentin and p-AKT proteins were decreased （P<0.05 or P<0.01）. Conclusion Over-expression of H2AX protein is closely associated with poor prognosis of the patients with PCa. The novel HDACi inhibitor CUDC-101 can regulate the phosphorylations of H2AX and AKT and inhibit the EMT process in the PCa cells.

Key words: CUDC-101, Histone H2A family member X, Prostate neoplasm, Epithelial-mesenchymal transition, DNA damage

中图分类号:

R737.2

那佈其,权春姬,赵芳,杨凡,肖茹,金雪梅,李珍玲. 组蛋白去乙酰化酶抑制剂CUDC-101对前列腺癌DU145细胞DNA损伤、迁移和上皮-间质转化的影响[J]. 吉林大学学报(医学版), 2024, 50(2): 400-410.

Buqi NA,Chunji QUAN,Fang ZHAO,Fan YANG,Ru XIAO,Xuemei JIN,Zhenling LI. Effect of histone deacetylase inhibitor CUDC-101 on DNA damage, migration, and epithelial-mesenchymal transition of prostate cancer DU145 cells[J]. Journal of Jilin University(Medicine Edition), 2024, 50(2): 400-410.

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Clinicapathological characteristic	n	H2AX protein expression		Strong positive rate（η/%）	χ²	P
Clinicapathological characteristic	n	－－+	?－?	Strong positive rate（η/%）	χ²	P
Age (year)
≤65	23	11	12	52.1	1.806	0.179
>65	106	35	71	66.9	1.806	0.179
Tumor stage
T1-T2	48	26	22	45.8	11.413	0.001
T3-T4	81	20	61	75.3	11.413	0.001
Gleason score
≤7	78	32	46	58.9	2.477	0.116
>7	51	14	37	72.5	2.477	0.116
WHO/ISUP
GG1	14	9	5	35.7	15.641	0.004
GG2	34	18	16	47.0
GG3 GG4 GG5	30 22 29	5 6 8	25 16 21	83.3 72.7 72.4
Lympnode metastasis
Yes	19	7	12	63.1	0.014	0.907
No	110	39	71	64.5	0.014	0.907
Perineural invasion
Yes	57	16	41	71.9	2.563	0.109
No	72	30	42	58.3	2.563	0.109
Vascular invasion
Yes	6	2	4	66.7	0.015	0.636
No	123	44	79	64.2	0.015	0.636

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