G-rich oligonucleotides inhibit HIF-1alpha and HIF-2alpha and block tumor growth.

Hypoxia-inducible factor-1 (HIF-1) plays crucial roles in tumor promotion by upregulating its target genes, which are involved in energy metabolism, angiogenesis, cell survival, invasion, metastasis, and drug resistance. The HIF-1alpha subunit, which is regulated by O2-dependent hydroxylation, ubiquitination, and degradation, has been identified as an important molecular target for cancer therapy. We have rationally designed G-rich oligodeoxynucleotides (ODNs) as inhibitors of HIF-1alpha for human cancer therapy. The lead compounds, JG243 and JG244, which form an intramolecular parallel G-quartet structure, selectively target HIF-1alpha and decreased levels of both HIF-1alpha and HIF-2alpha (IC50 < 2 micromol/l) and also inhibited the expression of HIF-1-regulated proteins [vascular endothelial growth factor (VEGF), Bcl-2, and Bcl-XL], but did not disrupt the expression of p300, Stat3, or p53. JG-ODNs induced proteasomal degradation of HIF-1alpha and HIF-2alpha that was dependent on the hydroxylase activity of prolyl-4-hydroxylase-2. JG243 and JG244 dramatically suppressed the growth of prostate, breast, and pancreatic tumor xenografts. Western blots from tumor tissues showed that JG-ODNs significantly decreased HIF-1alpha and HIF-2alpha levels and blocked the expression of VEGF. The JG-ODNs are novel anticancer agents that suppress tumor growth by inhibiting HIF-1.

A G-quartet oligonucleotide blocks glycoprotein Ib-mediated platelet adhesion and aggregation under flow conditions.

Platelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury. This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets. However, the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibalpha, the VWF-binding subunit of the GP Ib-IX-V complex. We evaluated the potential interactions of T30923 with GP Ibalpha and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the beta-sheets of the N-terminal region of GP Ibalpha (E14-D18, S39, D63-S64, and D83-S85). Experimentally, T30923 bound GP Ibalpha and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilised VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP Ibalpha-VWF interaction.

Computational study on mechanism of G-quartet oligonucleotide T40214 selectively targeting Stat3.

The mounting evidences have shown that signal transducer and activator of transcription 3 (Stat3) is a critical target for cancer therapy. Recently, we developed a G-quartet oligonucleotide T40214 as a novel and potent Stat3 inhibitor. T40214 specifically inhibited DNA-binding activity of Stat3 and significantly suppressed the growth of many tumor xenografts in nude mice. To determine the mechanism of GQ-ODNs selectively targeting Stat3, we established a 3D model of complex T40214/p-Stat3 dimer based on experimental evidences. The binding site of T40214 within Stat3 dimer was determined by statistical docking analysis. The results indicated that T40214 strongly interacted within the region from residue E638 through E652 of Stat3 dimer. The binding model refined by Hex docking disclosed that T40214 binds to SH2 domain of Stat3 and forms H-bonds with residues Q643, Q644, N646, and N647, which are critical for the binding interaction. The 3D models also suggested that T40214 inhibits Stat3 activity through disrupting the binding interaction between Stat3 dimer and DNA duplex for transcription. Our computational studies provided a platform for future structure-based drug design of novel Stat3 inhibitors.

Inhibition of Stat3 activation and tumor growth suppression of non-small cell lung cancer by G-quartet oligonucleotides.

Lung cancer is the leading cause of cancer mortality in the United States. Despite advances made over the past decades, the overall survival of patients with lung cancer remains dismal. Here we report novel G-quartet oligodeoxynucleotides (GQ-ODN) that were designed to selectively target signal transducer and activator of transcription 3 (Stat3), in the treatment of human non-small cell lung cancer (NSCLC). The objective of this study was to evaluate the effects of two novel GQ-ODN STAT3 inhibitors, T40214 and T40231, on NSCLC bearing nude mice. NSCLC bearing nude mice were assigned to 5 groups, which were treated by vehicle, control ODN, T40214, T40231, and Paclitaxel, respectively. Tumors were measured, isolated and analyzed using Western blotting, immuno-histochemistry, RPA and TUNEL. Results show that GQ-ODN T40214 and T40231 significantly suppress the growth of NSCLC tumors in nude mice by selectively inhibiting the activation of Stat3 and its downstream proteins Bcl-2, Bcl-xL, Mcl-1, survivin, VEGF, Cyclin D1 and c-myc; thereby, promoting apoptosis and reducing angiogenesis and cell proliferation. These findings validate Stat3 as an important molecular target for NSCLC therapy and demonstrate the efficacy of GQ-ODN in inhibiting Stat3 phosphorylation.

A novel mutation of the cytochrome-b5 reductase gene in an Indian patient: the molecular basis of type I methemoglobinemia.

We report here a novel mutation in the cytochrome b5 reductase gene resulting in type I methemoglobinemia. A single T->C transition in exon 8 at position 25985 was identified, changing codon 217 from Leu to Pro (L217P). The mutation is located in the NADH binding domain at the base of alpha-helix Nalpha3, a region of sequence highly conserved from yeast to man. A quantitative assessment of the thermodynamic cost of this mutation at 37 degrees C revealed a ten-fold drop in the free energy of stability. Alterations in hydrogen bonding and solvent accessibility surrounding residue 217 were predicted based on computer modeling.

Targeting signal transducer and activator of transcription 3 with G-quartet oligonucleotides: a potential novel therapy for head and neck cancer.

Signal transducer and activator of transcription 3 (Stat3) is a critical mediator of oncogenic signaling activated frequently in many types of human cancer where it contributes to tumor cell growth and resistance to apoptosis. Stat3 has been proposed as a promising target for anticancer drug discovery. Recently, we developed a series of G-quartet oligodeoxynucleotides (GQ-ODN) as novel and potent Stat3 inhibitors, which significantly suppressed the growth of prostate and breast tumors in nude mice. In the present study, we showed that GQ-ODN specifically inhibited DNA-binding activity of Stat3 as opposed to Stat1. Computer-based docking analysis revealed that GQ-ODN predominantly interacts with the SH2 domains of Stat3 homodimers to destabilize dimer formation and disrupt DNA-binding activity. We employed five regimens in the treatment of nude mice with tumors of head and neck squamous cell carcinoma (HNSCC): placebo, paclitaxel, GQ-ODN T40214, GQ-ODN T40231, and T40214 plus paclitaxel. The mean size of HNSCC tumors over 21 days only increased by 1.7-fold in T40214-treated mice and actually decreased by 35% in T40214 plus paclitaxel-treated mice whereas the mean size of HNSCC tumors increased 9.4-fold in placebo-treated mice in the same period. These findings show that GQ-ODN has potent activity against HNSCC tumor xenografts alone and in combination with paclitaxel.