Effect of Base Sequence on G-Wire Formation in Solution

The formation and dimensions of G-wires by different short G-rich DNA sequences in solution were investigated by dynamic light scattering (DLS) and polyacrilamide gel electrophoresis (PAGE). To explore the basic principles of wire formation, we studied the effects of base sequence, method of preparation, temperature, and oligonucleotide concentration. Both DLS and PAGE show that thermal annealing induces much less macromolecular self-assembly than dialysis. The degree of assembly and consequently length of G-wires (5-6 nm) are well resolved by both methods for DNA sequences with intermediate length, while some discrepancies appear for the shortest and longest sequences. As expected, the longest DNA sequence gives the longest macromolecular aggregates with a length of about 11 nm as estimated by DLS. The quadruplex topologies show no concentration dependence in the investigated DNA concentration range (0.1 mM–0.4 mM) and no structural change upon heating

Kinetics of double-chain reversals bridging contiguous quartets in tetramolecular quadruplexes

Repetitive 5′GGXGG DNA segments abound in, or near, regulatory regions of the genome and may form unusual structures called G-quadruplexes. Using NMR spectroscopy, we demonstrate that a family of 5′GCGGXGGY sequences adopts a folding topology containing double-chain reversals. The topology is composed of two bistranded quadruplex monomeric units linked by formation of G:C:G:C tetrads. We provide a complete thermodynamic and kinetic analysis of 13 different sequences using absorbance spectroscopy and DSC, and compare their kinetics with a canonical tetrameric parallel-stranded quadruplex formed by TG(4)T. We demonstrate large differences (up to 10(5)-fold) in the association constants of these quadruplexes depending on primary sequence; the fastest samples exhibiting association rate equal or higher than the canonical TG(4)T quadruplex. In contrast, all sequences studied here unfold at a lower temperature than this quadruplex. Some sequences have thermodynamic stability comparable to the canonical TG(4)T tetramolecular quadruplex, but with faster association and dissociation. Sequence effects on the dissociation processes are discussed in light of structural data

Crocin Inhibits Angiogenesis and Metastasis in Colon Cancer via TNF-α/NF-kB/VEGF Pathways

Angiogenesis and metastasis play pivotal roles in the progression of cancer. We recently discovered that crocin, a dietary carotenoid derived from the Himalayan crocus, inhibited the growth of colon cancer cells. However, the exact role of crocin on the angiogenesis and metastasis in colorectal cancer remains unclear. In the present study, we demonstrated that crocin significantly reduces the viability of colon cancer cells (HT-29, Caco-2) and human umbilical vein endothelial cells (HUVEC), but was not toxic to human colon epithelial (HCEC) cells. Furthermore, pre-treatment of human carcinoma cells (HT-29 and Caco-2) with crocin inhibited cell migration, invasion, and angiogenesis in concentration -dependent manner. Further studies demonstrated that crocin inhibited TNF-α, NF-κB and VEGF pathways in colon carcinoma cell angiogenesis and metastasis. Crocin also inhibited cell migration, invasion, and tube formation in human umbilical vein endothelial cells (HUVEC) in a concentration -dependent manner. We also observed that crocin significantly reduced the secretion of VEGF and TNF-α induced activation of NF-kB by human colon carcinoma cells. In the absence of TNF-α, a concentration-dependent reduction in NF-kB was observed. Many of these observations were confirmed by in vivo angiogenesis models, which showed that crocin significantly reduced the progression of tumour growth. Collectively, these finding suggest that crocin inhibits angiogenesis and colorectal cancer cell metastasis by targeting NF-kB and blocking TNF-α/NF-κB/VEGF pathways

How long is too long? Effects of loop size on G-quadruplex stability

We compared here 80 different sequences containing four tracts of three guanines with loops of variable length (between 1 and 15 bases for unmodified sequences, up to 30 for fluorescently labeled oligonucleotides). All sequences were capable of forming stable quadruplexes, with Tm above physiological temperature in most cases. Unsurprisingly, the melting temperature was systematically lower in sodium than in potassium but the difference between both ionic conditions varied between 1 and >39°C (average difference: 18.3°C). Depending on the sequence context, and especially for G4 sequences involving two very short loops, the third one may be very long without compromising the stability of the quadruplex. A strong inverse correlation between total loop length and Tm was found in K+: each added base leads to a 2°C drop in Tm or ∼0.3 kcal/mol loss in ΔG°. The trend was less clear in Na+, with a longer than expected optimal loop length (up to 5 nt). This study will therefore extend the sequence repertoire of quadruplex-prone sequences, arguing for a modification of the widely used consensus (maximal loop size of 7 bases)

Conformational changes in quadruplex oligonucleotide structures probed by Raman spectroscopy

Quadruplex structures are higher order structures formed by guanine-rich oligonucleotides. In the present study, temperature-induced conformational changes in the quadruplex structures of aptamers and other guanine-rich oligonucleotides are probed by Raman spectroscopy. In particular, dramatic changes in the fingerprint region are observed in the spectra of thrombin binding aptamer at higher temperatures. These changes are accompanied by a decrease in the intensity of the 1480 cm−1 peak (attributed to C8 = N7-H2), which is diagnostic of the quadruplex structure. We also show that these changes can be reversed (to a certain extent) by addition of K+ ions

Albumin Nano-Encapsulation of Piceatannol Enhances Its Anticancer Potential in Colon Cancer Via Downregulation of Nuclear p65 and HIF-1 alpha

Piceatannol (PIC) is known to have anticancer activity, which has been attributed to its ability to block the proliferation of cancer cells via suppression of the NF-kB signaling pathway. However, its effect on hypoxia-inducible factor (HIF) is not well known in cancer. In this study, PIC was loaded into bovine serum albumin (BSA) by desolvation method as PIC-BSA nanoparticles (NPs). These PIC-BSA nanoparticles were assessed for in vitro cytotoxicity, migration, invasion, and colony formation studies and levels of p65 and HIF-1α. Our results indicate that PIC-BSA NPs were more effective in downregulating the expression of nuclear p65 and HIF-1α in colon cancer cells as compared to free PIC. We also observed a significant reduction in inflammation induced by chemical colitis in mice by PIC-BSA NPs. Furthermore, a significant reduction in tumor size and number of colon tumors was also observed in the murine model of colitis-associated colorectal cancer, when treated with PIC-BSA NPs as compared to free PIC. The overall results indicate that PIC, when formulated as PIC-BSA NPs, enhances its therpautice potential. Our work could prompt further research in using natural anticancer agents as nanoparticels with possiable human clinical trails. This could lead to the development of a new line of safe and effective therapeutics for cancer patients

Dietary Crocin is Protective in Pancreatic Cancer while Reducing Radiation-Induced Hepatic Oxidative Damage

Pancreatic cancer is one of the fatal causes of global cancer-related deaths. Although surgery and chemotherapy are standard treatment options, post-treatment outcomes often end in a poor prognosis. In the present study, we investigated anti-pancreatic cancer and amelioration of radiation-induced oxidative damage by crocin. Crocin is a carotenoid isolated from the dietary herb saffron, a prospect for novel leads as an anti-cancer agent. Crocin significantly reduced cell viability of BXPC3 and Capan-2 by triggering caspase signaling via the downregulation of Bcl-2. It modulated the expression of cell cycle signaling proteins P53, P21, P27, CDK2, c-MYC, Cyt-c and P38. Concomitantly, crocin treatment-induced apoptosis by inducing the release of cytochrome c from mitochondria to cytosol. Microarray analysis of the expression signature of genes induced by crocin showed a substantial number of genes involved in cell signaling pathways and checkpoints (723) are significantly affected by crocin. In mice bearing pancreatic tumors, crocin significantly reduced tumor burden without a change in body weight. Additionally, it showed significant protection against radiation-induced hepatic oxidative damage, reduced the levels of hepatic toxicity and preserved liver morphology. These findings indicate that crocin has a potential role in the treatment, prevention and management of pancreatic cancer

Resolution and characterization of the structural polymorphism of a single quadruplex-forming sequence

The remarkable structural polymorphism of quadruplex-forming sequences has been a considerable impediment in the elucidation of quadruplex folds. Sequence modifications have commonly been used to perturb and purportedly select a particular form out of the ensemble of folds for nuclear magnetic resonance (NMR) or X-ray crystallographic analysis. Here we report a simple chromatographic technique that separates the individual folds without need for sequence modification. The sequence d(GGTGGTGGTGGTTGTGGTGGTGGTGG) forms a compact quadruplex according to a variety of common biophysical techniques. However, NMR and chromatography showed that this oligonucleotide produces at least eight monomeric quadruplex species that interconvert very slowly at room temperature. We have used a combination of spectroscopic, hydrodynamic and thermodynamic techniques to evaluate the physicochemical properties of the mixture and the individual species. These species have almost identical thermodynamic, hydrodynamic and electrophoretic properties, but significantly different NMR and circular dichroism (CD) spectra, as well as kinetic stability. These results demonstrate that simple standard low-resolution techniques cannot always be used for quadruplex fold determination or quality control purposes, and that simple thermodynamic analysis does not directly provide interpretable thermodynamic parameters