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Aristolochic acid I interferes with the expression of BLCAP tumor suppressor gene in human cells
Ying-Tzu Huang1, Ting-Shuan Wu1, Chuan-Chen Lu2
1Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Abstract:
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.
Insights
Aristolochic acid I (AAI) reduces bladder cancer-associated protein (BLCAP) expression, contributing to cell malignancy. Restoring BLCAP expression may counteract AAI-induced carcinogenesis.
Area of Science:
- Toxicology
- Molecular Biology
- Oncology
Background:
- Aristolochic acid I (AAI) is a phytocompound implicated in renal disease and urothelial carcinoma.
- The bladder cancer-associated protein (BLCAP) gene functions as a tumor suppressor in various cancers, including bladder cancer.
Purpose of the Study:
- To investigate the effect of Aristolochic acid I (AAI) on BLCAP gene expression.
- To elucidate the underlying mechanisms by which AAI influences BLCAP expression in human cells.
Main Methods:
- Administered AAI to human embryonic kidney (HEK293), proximal tubule epithelial (HK-2), and bladder cancer (HT-1376) cells.
- Assessed BLCAP mRNA and protein levels, BLCAP promoter activity via luciferase assays, and transcription factor activities (AP-1, NF-κB).
- Utilized DNA methyltransferase inhibitor (5-aza-dC), methyl-specific PCR, and bisulfate sequencing to explore methylation effects; performed BLCAP knockdown to assess functional consequences.
Main Results:
- AAI significantly decreased BLCAP mRNA and protein expression across all tested cell lines.
- AAI suppressed BLCAP promoter activity and reduced AP-1/NF-κB activities, though these sites were not directly responsible for AAI's effect.
- 5-aza-dC treatment reversed AAI-induced BLCAP downregulation, indicating a role for epigenetic modifications, yet AAI did not alter BLCAP promoter methylation.
- BLCAP knockdown enhanced cellular migration, invasion, proliferation, and anchorage-independent growth, suggesting a tumor-suppressive role.
Conclusions:
- AAI downregulates BLCAP gene expression through mechanisms not directly involving AP-1/NF-κB binding sites or promoter hypermethylation.
- The observed reduction in BLCAP expression by AAI contributes to cellular malignant transformation.
- BLCAP deficiency may play a significant role in mediating Aristolochic acid-associated carcinogenesis.
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