A novel small molecule inhibitor of the DNA repair protein Ku70/80.

Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.

Induction of DNA damage in human urothelial cells by the brominated flame retardant 2,2-bis(bromomethyl)-1,3-propanediol: role of oxidative stress.

2,2-bis(bromomethyl)-1,3-propanediol (BMP) is an extensively used brominated flame retardant found in urethane foams and polyester resins. In a 2-year dietary study conducted by the National Toxicology Program, BMP caused neoplastic lesions at multiple sites including the urinary bladder in both rats and mice. The mechanism of its carcinogenic effect is unknown. In the present study, using SV-40 immortalized human urothelial cells (UROtsa), endpoints associated with BMP induced DNA damage and oxidative stress were investigated. The effects of time (1-24h) and concentration (5-100 µM) on BMP induced DNA strand breaks were assessed via the alkaline comet assay. The results revealed evidence of DNA strand breaks at 1 and 3h following incubation of cells with non-cytotoxic concentrations of BMP. Strand breaks were not present after 6h of incubation. Evidences for BMP associated oxidative stress include: an elevation of intracellular ROS formation as well as induction of Nrf2 and HSP70 protein levels. In addition, DNA strand breaks were attenuated when cells were pre-treated with N-acetyl-l-cysteine (NAC) and oxidative base modifications were revealed when a lesion specific endonuclease, human 8-hydroxyguanine DNA glycosylase 1 (hOGG1) was introduced into the comet assay. In conclusion, these results demonstrate that BMP induces DNA strand breaks and oxidative base damage in UROtsa cells. Oxidative stress is a significant, determinant factor in mediating these DNA lesions. These early genotoxic events may, in part, contribute to BMP-induced carcinogenesis observed in rodents.

cAMP-dependent cytosolic mislocalization of p27(kip)-cyclin D1 during quinol-thioether-induced tuberous sclerosis renal cell carcinoma.

The loss of tuberin, the tuberous sclerosis-2 (Tsc-2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc-2(EK/+)) and in human metastatic renal cell carcinoma tissue. Signaling associated with cytoplasmic mislocalization of p27 in renal cancer is relatively unknown. Renal tumors derived from 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-treated Tsc-2(EK/+) rats, and null for tuberin, display elevated nuclear and cytosolic p27, with parallel increases in cytosolic cyclin D1 levels. Similar changes are observed in TGHQ-transformed renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), which, in addition to the cytoplasmic mislocalization of p27 and cyclin D1, exhibit high ERK, B-Raf, and Raf-1 kinase activity. Renal tumor xenografts, derived from subcutaneous injection of QTRRE cells into nude mice, also display increases in cytosolic mislocalization of p27 and cyclin D1. Dibutyryl cAMP and/or phosphodiesterase inhibitors (PIs; pentoxifylline or theophylline) increase Rap1B activation, B-Raf kinase activity, and cytosolic p27/cyclin D1 protein levels in QTRRE cells. Inhibition of Raf kinases with either sorafenib or B-Raf small interfering RNA (siRNA) caused a mitogen-activated protein kinase-mediated downregulation of p27. Moreover, decreases in cyclin D1 were also associated with p27 siRNA knockdown in QTRRE cells. Finally, theophylline-mediated increases in p27 and cyclin D1 were attenuated by sorafenib, which modulated Raf/MEK/ERK signaling. Collectively, these data suggest that the cAMP/Rap1B/B-Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27-cyclin D1 in tuberous sclerosis gene-regulated-renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27-cyclin D1 cytosolic stabilization during renal tumor formation.

Identification of thioredoxin-interacting protein 1 as a hypoxia-inducible factor 1alpha-induced gene in pancreatic cancer.

OBJECTIVE: To investigate the expression of thioredoxin-interacting protein (TXNIP) during hypoxia and its dependency on hypoxia-inducible factor 1alpha (HIF-1alpha) in pancreatic cancer cell lines. METHODS: MiaPaCa-2 pancreatic cancer cells were transiently transfected with siRNA to HIF-1alpha and TXNIP protein measured after growth in normoxia or hypoxia. In addition, HIF-1alpha dependency was assessed by transiently transfecting MiaPaCa-2 pancreatic cancer cells with HIF-1alpha with a mutated oxygen degradation domain resulting in stable HIF-1alpha expression in normoxic conditions. Panc-1 pancreatic cancer cells with low endogenous TXNIP expression were stably transfected with TXNIP, and cell survival and response to platinum cancer agents were tested. Quantitative immunohistochemistry was utilized to measure the expression of TXNIP and thioredoxin 1 in human pancreatic cancer tissues. RESULTS: Thioredoxin-interacting protein was induced during hypoxia in pancreatic cancer cells in a HIF-1alpha-dependent manner. Overexpression of TXNIP in the Panc-1 cells resulted in a higher basal apoptosis and increased sensitivity to cisplatin and oxaliplatin. A negative correlation was observed between TXNIP and thioredoxin 1 expression in human pancreatic cancer tissues. CONCLUSIONS: Thioredoxin-interacting protein, a putative tumor suppressor gene, is induced in response to hypoxia in a HIF-1alpha-dependent manner in pancreatic cancer cells, resulting in increased apoptosis and increased sensitivity to platinum anticancer therapy. Increased TXNIP may be a mechanism to counterbalance the prosurvival effects of HIF-1alpha.

Increased expression of thioredoxin-1 in human colorectal cancer is associated with decreased patient survival.

Thioredoxin-1 is a redox protein that, when overexpressed, causes increased cancer-cell growth and inhibited apoptosis. Thioredoxin-1 expression has been reported to be increased in several human primary tumors, but its relationship to tumor progression and patient survival has not been established. We studied the expression of thioredoxin-1 as measured with immunohistochemical staining in paraffin-embedded human normal colonic mucosa, adenomatous polyps, and primary and metastatic colorectal cancer. Thioredoxin-1 expression was not increased in 12 colorectal adenomatous polyps, compared with 8 samples of normal colonic mucosa, but was significantly increased in 12 primary colorectal cancers (P <.01). Thioredoxin-1 expression was not significantly different in primary lymph-node metastases and the primary colorectal cancer. Using colorectal cancer samples from 37 subjects for whom survival data was available, we found that thioredoxin-1 expression increased with Dukes stage, although the association was not statistically significant (P =.077). We noted a significant association between thioredoxin-1 expression and patient survival (P =.004); higher score was associated with decreased survival. When adjusted for Dukes stage, thioredoxin-1 expression showed a statistically significant association with survival (P =.012). The work shows that increased thioredoxin-1 expression is a relatively late event in colorectal carcinogenesis and provides evidence in a small group of subjects with colorectal cancer of Dukes stages A through D that thioredoxin-1 expression may be an independent marker of patient prognosis.