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赵亚楠, 陈利弘, 杨晓龙, 等. 四硫代钼酸铵抑制铜转运蛋白-1用于胰腺癌治疗的体内外研究[J]. 四川大学学报(医学版), 2020, 51(5): 643-649. DOI: 10.12182/20200960101
引用本文: 赵亚楠, 陈利弘, 杨晓龙, 等. 四硫代钼酸铵抑制铜转运蛋白-1用于胰腺癌治疗的体内外研究[J]. 四川大学学报(医学版), 2020, 51(5): 643-649. DOI: 10.12182/20200960101
ZHAO Ya-nan, CHEN Li-hong, YANG Xiao-long, et al. Inhibition of Copper Transporter-1 by Ammonium Tetrathiocarbolybdate in the Treatment of Pancreatic Cancer[J]. Journal of Sichuan University (Medical Sciences), 2020, 51(5): 643-649. DOI: 10.12182/20200960101
Citation: ZHAO Ya-nan, CHEN Li-hong, YANG Xiao-long, et al. Inhibition of Copper Transporter-1 by Ammonium Tetrathiocarbolybdate in the Treatment of Pancreatic Cancer[J]. Journal of Sichuan University (Medical Sciences), 2020, 51(5): 643-649. DOI: 10.12182/20200960101

四硫代钼酸铵抑制铜转运蛋白-1用于胰腺癌治疗的体内外研究

Inhibition of Copper Transporter-1 by Ammonium Tetrathiocarbolybdate in the Treatment of Pancreatic Cancer

  • 摘要:
      目的  检测胰腺癌细胞、原位异种移植的原位胰腺癌小鼠模型和临床胰腺癌组织标本中铜转运蛋白1(copper transporter 1, CTR1)的表达,并验证铜螯合剂四硫代钼酸铵(ammonium tetrathiomolybdate, TM)通过调控胰腺癌细胞中CTR1的表达对胰腺癌的抑制效果。
      方法  采用免疫组化法检测22例临床胰腺导管癌及距肿瘤0.5~1 cm的癌旁组织标本中CTR1和抗氧化蛋白1(ATOX1)的表达情况。采用PANC-1细胞构建5只原位胰腺癌裸鼠模型,收集胰腺癌组织中及对应的正常胰腺组织,免疫组化法检测CTR1和ATOX1的表达情况,与临床标本进行比较。采用10、30、50、100 μmol/L TM处理人胰腺癌细胞株PANC-1 24 h、48 h、72 h后,使用CCK-8法检测细胞增殖情况;50 μmol/L TM处理PANC-1 24 h、48 h后,划痕实验检测PANC-1细胞侵袭能力变化;10、30、50、100 μmol/L TM处理PANC-1 48 h后,Western blot检测CTR1、血管内皮生长因子(vascular endothelial growth factor, VEGF)和细胞周期蛋白CyclinD1的表达。采用PANC-1细胞建立裸鼠皮下荷瘤模型,分别给予不同剂量TM(0.3 mg/d,1.0 mg/d)灌胃24 d后,观察肿瘤生长情况。
      结果  免疫组化结果提示,22例临床胰腺导管癌患者组织标本中,19例(86.36%)出现CTR1高表达,且在PANC-1裸鼠原位移植瘤组织中CTR1同样高表达。使用TM处理PANC-1细胞后,细胞增殖抑制程度随着TM剂量的升高、处理时间的延长而逐渐增强;细胞迁移能力下降;胞内CTR1、VEGF和CyclinD1的表达均随着TM剂量的升高而降低。PANC-1荷瘤裸鼠在给予不同剂量TM后,肿瘤生长出现抑制,TM高剂量组的肿瘤体积和肿瘤质量减小更为明显(P<0.05)。
      结论  胰腺癌中CTR1表达异常增高,针对这一靶点使用铜螯合剂治疗可能有助于抑制胰腺癌生长迁移。

     

    Abstract:
      Objective  To examine copper transporter 1 (CTR1) expression in pancreatic carcinoma cells, orthotopic xenograft pancreatic tumor model and clinical samples, and verify the effect of copper chelating agent ammonium tetrathiomolybdate (TM) regulate the expression of CTR1 in pancreatic carcinoma cells and the inhibition of pancreatic carcinoma.
      Methods  The expressions of copper transporter CTR1 and antioxidant protein 1 (ATOX1) in 22 clinical pancreatic ductal carcinoma and paracancer tissues 0.5-1 cm away from the tumor were measured by immunohistochemistry (IHC). PANC-1 cells were used to construct 5 orthotopic xenograft pancreatic tumor of nude mice models. Pancreatic cancer tissues and corresponding normal pancreatic tissues were collected, and the expressions of CTR1 and ATOX1 were detected by IHC and compared with clinical tissues. The proliferation of pancreatic carcinoma cells PANC-1 treated with 10, 30, 50, 100 μmol/L TM for 24 h, 48 h, 72 h was measured by CCK8 assay. The migration abilities of PANC-1 cells treated with 50 μmol/L TM for 24 h, 48 h were detected by scratch test. The expressions of CTR1, vascular endothelial growth factor (VEGF) and CyclinD1 proteins in PANC-1 cells treated with 10, 30, 50, 100 μmol/L TM for 48 h were measured by Western blot. Then the subcutaneous tumor-bearing model of nude mice were established with PANC-1 cells, and the growth of tumor was observed after oral administration of 0.3 mg/d and 1.0 mg/d of TM, respectively.
      Results  The immunohistochemical results indicated that 19 of the 22 clinical pancreatic ductal cancer tissues of carcinoma patients had high expression of CTR1, and the same high expression of CTR1 was found in the orthotopic transplanted tumor tissues of PANC-1 nude mice. The proliferation inhibition of PANC-1 cells increased with the concentration of TM increased and the treatment time prolonged. The expressions of intracellular CTR1, VEGF and CyclinD1 all decreased with the concentration of TM increased. The cell migration ability decreased after the PANC-1 cells treated with TM. The tumor growth of PANC-1 tumor-bearing nude mice was inhibited after different doses of TM were delivered. The reduction in tumor volume and weight was more pronounced in the high-dose TM group (P<0.05).
      Conclusion  The expression of CTR1 is abnormally elevated in pancreatic carcinoma, and treatment with copper chelating agent for this target may help to inhibit pancreatic carcinoma.

     

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