Gene expression profiles modulated by the human carcinogen aristolochic acid I in human cancer cells and their dependence on TP53

Abstract

Aristolochic acid (AA) is the causative agent of urothelial tumours associated with aristolochic acid nephropathy. These tumours contain TP53 mutations and over-express TP53. We compared transcriptional and translational responses of two isogenic HCT116 cell lines, one expressing TP53 (p53-WT) and the other with this gene knocked out (p53-null), to treatment with aristolochic acid I (AAI) (50−100 µM) for 6−48 h. Modulation of 118 genes was observed in p53-WT cells and 123 genes in p53-null cells. Some genes, including INSIG1, EGR1, CAV1, LCN2 and CCNG1, were differentially expressed in the two cell lines. CDKN1A was selectively up-regulated in p53-WT cells, leading to accumulation of TP53 and CDKN1A. Apoptotic signalling, measured by caspase-3 and -7 activity, was TP53-dependent. Both cell types accumulated in S phase, suggesting that AAI-DNA adducts interfere with DNA replication, independently of TP53 status. The oncogene MYC, frequently over-expressed in urothelial tumours, was up-regulated by AAI, whereas FOS was downregulated. Observed modulation of genes involved in endocytosis, e.g. RAB5A, may be relevant to the known inhibition of receptor-mediated endocytosis, an early sign of AA-mediated proximal tubule injury. AAI-DNA adduct formation was significantly greater in p53-WT cells than in p53-null cells. Collectively, phenotypic anchoring of the AAI-induced expression profiles to DNA adduct formation, cell-cycle parameters, TP53 expression and apoptosis identified several genes linked to these biological outcomes, some of which are TP53-dependent. These results strengthen the importance of TP53 in AA-induced cancer, and indicate that other alterations, e.g. to MYC oncogenic pathways, may also contribute.

Aristolochic acid suppresses DNA repair and triggers oxidative DNA damage in human kidney proximal tubular cells

“…CDKN1A mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress signals, including genotoxic stress, hypoxia, and oncogene activation. Our results were opposite to those reported (35) in p53-null cells after a 48-h exposure to AA (100 μM) in HCT 116 cell line. Previous data from Chang (33) showed that AA mixture (AAM; 41% AA I, 56% AA II) can cause arrest in the G0/G1 phase (from 37.6 to 49.2%) in human urinary tract epithelium cells (SV-HUC-1), suggesting that AA-induced cell cycle arrest at the G0/G1 phase is associated with cyclin E/cdk2 complex (the cdk inhibitor).…”

“…Several studies showed that AA can induce cytotoxic effects in human colon cancer HCT 116 cells, porcine renal LLC-PK1 cells, and opossum kidney (OK) cell line. The HK-2 cells used in our study were more sensitive to the cytotoxicity of AA than HCT 116 cells (35), and OK cells (40). The IC 50 of 24 h AA treatment for HK-2 cells and HCT 116 cells was 30 and 100 μM, respectively.…”

“…Balachandran et al (31) and Li et al (32) have used a porcine proximal tubular epithelial cell line (LLC-PK1) to study DNA damage, cell cycle perturbations, and cell apoptosis induced by AA. Human urinary tract epithelium cells (SV-HUC-1), human hepatoma HepG2 cells, and human colorectal cancer cells (HCT 116) (33)(34)(35) have also been used to study the cytotoxicity or genotoxicity of AA. Chen et al (36) have shown that more cancer-related genes were significantly altered in target (kidney) tissues than in non-target (liver) tissues in AA-treated rats, suggesting that studies on the toxic effects of AA should be conducted in renal cells.…”

Microarray analysis reveals the inhibition of nuclear factor-kappa B signaling by aristolochic acid in normal human kidney (HK-2) cells

“…The IC 50 values of AA after 24 h treatment in HK-2 cells and HCT 116 cells were 30 μmol/L and 100 μmol/L, respectively. Moreover, the effect of AA on gene expression was more profound in HK-2 cells than in HCT 116 cells [27] . A previous report indicated no significant difference in TP53 expression in P53-WT HCT 116 cells after a 24 h exposure to AA (100 μmol/L), whereas our study showed a marked downregulation of TP53 gene expression (fold change -1.92 after qRT-PCR confirmation) in 10 μmol/L AA-treated HK-2 cells (data not shown).…”

“…In this in vitro study on the gene expression profiles of AA-treated (HK-2) cells, the normal human proximal tubular cell line was used, in contrast with the human colorectal cancer cell line (HCT 116) used by Simoes [27] . Our results showed that HK-2 cells were more sensitive to AA-induced cytotoxicity than HCT 116 cells.…”