Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan.
Toxicol Lett. 2018 Jul;291:129-137. doi: 10.1016/j.toxlet.2018.03.032. Epub 2018 Apr 12.
Aristolochic acid I (AAI) is a phytocompound that is linked to the progressive renal disease and development of human urothelial carcinoma. The bladder cancer-associated protein (BLCAP) gene exhibits a tumor suppressor function in various tumors, including bladder carcinoma. This study evaluated the effect of AAI on BLCAP expression and its associated mechanism in human cells. Administering AAI to human embryonic kidney cells (HEK293), human proximal tubule epithelial cells (HK-2) and urinary bladder cancer cells (HT-1376) significantly reduced the expression of BLCAP mRNA and protein. AAI also effectively suppressed the luciferase activities driven by BLCAP promoters of various lengths in HEK293 cells. AAI significantly reduced both activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities in reporter assays, but further point mutations revealed that Ap-1 and NF-κB binding sites on the BLCAP promoter were not AAI-responsive elements. Application of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), reversed the decline of BLCAP expression that had been induced by AAI. However, AAI exposure did not alter hypermethylation of the BLCAP promoter, determined by methyl-specific polymerase chain reaction (PCR) and bisulfate sequencing. Knocking down BLCAP in HEK293 cell line enhanced the potential for cellular migration, invasion, and proliferation, along with the induction of a capacity for anchorage-independent growth. In conclusion, AAI down-regulated the expression of BLCAP gene and the deficiency in BLCAP expression contributed to the malignant transformation of human cells, implying that BLCAP may have a role in mediating AAI-associated carcinogenesis.
马兜铃酸 I（AAI）是一种植物化合物，与进行性肾疾病和人类尿路上皮癌的发展有关。膀胱癌相关蛋白（BLCAP）基因在多种肿瘤中具有肿瘤抑制功能，包括膀胱癌。本研究评估了 AAI 对人细胞中 BLCAP 表达的影响及其相关机制。在人胚肾细胞（HEK293）、人近端肾小管上皮细胞（HK-2）和膀胱癌细胞（HT-1376）中给予 AAI，可显著降低 BLCAP mRNA 和蛋白的表达。AAI 还能有效抑制 HEK293 细胞中各种长度的 BLCAP 启动子驱动的荧光素酶活性。AAI 可显著降低报告基因检测中的激活蛋白 1（AP-1）和核因子-κB（NF-κB）活性，但进一步的点突变表明，BLCAP 启动子上的 Ap-1 和 NF-κB 结合位点不是 AAI 反应元件。DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷（5-aza-dC）的应用可逆转 AAI 诱导的 BLCAP 表达下降。然而，AAI 暴露并没有改变 BLCAP 启动子的高甲基化，这是通过甲基特异性聚合酶链反应（PCR）和亚硫酸氢盐测序确定的。在 HEK293 细胞系中敲低 BLCAP 可增强细胞迁移、侵袭和增殖的潜能，并诱导锚定非依赖性生长的能力。总之，AAI 下调 BLCAP 基因的表达，BLCAP 表达的缺失促进了人细胞的恶性转化，这表明 BLCAP 可能在介导 AAI 相关致癌作用中发挥作用。
