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.

Correction: Aljabali, A.A.A.; et al. Albumin Nano-Encapsulation of Piceatannol Enhances Its Anticancer Potential in Colon Cancer via down Regulation of Nuclear p65 and HIF-1α. Cancers 2020, 12, 113.

The authors wish to make the following corrections to this paper [...].

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.

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

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.

Encoding canonical DNA quadruplex structure.

The main challenge in DNA quadruplex design is to encode a three-dimensional structure into the primary sequence, despite its multiple, repetitive guanine segments. We identify and detail structural elements describing all 14 feasible canonical quadruplex scaffolds and demonstrate their use in control of design. This work outlines a new roadmap for implementation of targeted design of quadruplexes for material, biotechnological, and therapeutic applications.

Surface-Adsorbed Long G-Quadruplex Nanowires Formed by G:C Linkages.

G-quadruplexes connected into long, continuous nanostructures termed G-wires show properties superior to dsDNA when applied in nanotechnology. Using AFM imaging, we systematically studied surface adsorption of a set of G-rich oligonucleotides with GC-termini for their ability to form long G-wires through G:C pairing. We investigated the effects of increasing sequence length, the type of nucleotide in the side loops, and removal of the CG-3' terminus. We found that sequences with adenine in the side loops most readily form G-wires. The role of magnesium as an efficient surface-anchoring ion was also confirmed. Conversely, as resolved from dynamic light scattering measurements, magnesium had no ability to promote G-quadruplex formation in solution. These insights may help in selecting prosperous candidates for construction of G-quadruplex based nanowires and to explore them for their electronic properties.

DNA quadruplex folding formalism--a tutorial on quadruplex topologies.

Quadruplexes of DNA adopt a large variety of topologies that are dependent on their environment. We have been developing a formalism for quadruplex folding based on the relationship between base and its sugar--as defined by the glycosidic bond angle. By reducing the quadruplex stem to a description based on two finite states of the range of angles the glycosidic bond angle may adopt, the description of the relationships of type of loop and groove widths of a quadruplex stem are possible. In its current form this formalism has allowed for the prediction of some unimolecular quadruplex topologies. Its rules, whilst developed for unimolecular quadruplexes of three loops, are of general utility in understanding the interdependency of structural characteristics of multimolecular folds, as well as unimolecular quadruplexes of more than three loops. Here we describe current understanding of the interdependent structural features that define the quadruplex fold, and provide a tutorial for the use and application of this formalism.

Structural probes in quadruplex nucleic acid structure determination by NMR.

Traditionally, isotope-labelled DNA and RNA have been fundamental to nucleic acid structural studies by NMR. Four-stranded nucleic acid architectures studies increasingly benefit from a plethora of nucleotide conjugates for resonance assignments, the identification of hydrogen bond alignments, and improving the population of preferred species within equilibria. In this paper, we review their use for these purposes. Most importantly we identify reasons for the failure of some modifications to result in quadruplex formation.

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.

Geometric formalism for DNA quadruplex folding.

Understanding the control of self-assembly and stereochemical properties of DNA higher order architectural folds is of fundamental importance in biology as well as biochemical technological applications. Guanine-rich DNA sequences can form tetrahelical architectures termed quadruplexes. A formalism is presented describing the interdependency of a set of structural descriptors as a geometric basis for folding of unimolecular quadruplex topologies. It represents a standard for interpretation of structural characteristics of quadruplexes, and is comprehensive in explicitly harmonizing the results of published literature with a unified language. The formalism is a fundamental step towards prediction of unimolecular quadruplex folding topologies from primary sequence.

NMR methods for studying quadruplex nucleic acids.

Solution NMR spectroscopy has traditionally played a central role in examining quadruplex structure, dynamics, and interactions. Here, an overview is given of the methods currently applied to structural, dynamics, thermodynamics, and kinetics studies of nucleic acid quadruplexes and associated cations.

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 TG4T. 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 TG4T quadruplex. In contrast, all sequences studied here unfold at a lower temperature than this quadruplex. Some sequences have thermodynamic stability comparable to the canonical TG4T tetramolecular quadruplex, but with faster association and dissociation. Sequence effects on the dissociation processes are discussed in light of structural data.

Structure, flexibility, and repair of two different orientations of the same alkyl interstrand DNA cross-link.

Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeutic bifunctional alkylating agents. Using an N(4)C-ethyl-N(4)C interstrand DNA cross-link to mimic this class of clinically important cancer chemotherapeutic agents, we have characterized the repair, structure, and flexibility of DNA that contains this cross-link in two different orientations. Plasmid DNAs in which the cytosines of single CpG or GpC steps are covalently linked were efficiently processed by repair proficient and homologous recombination deficient strains of Escherichia coli. Repair in a nucleotide excision repair (NER) deficient strain was less efficient overall and displayed a 4-fold difference between the two cross-link orientations. Both the structure and flexibility of DNA containing these cross-links were examined using a combination of (1)H NMR, restrained molecular dynamics simulations, and atomic force microscopy (AFM). The NMR structure of a decamer containing a CpG interstrand cross-link shows the cross-link easily accommodated within the duplex with no disruption of hydrogen bonding and only minor perturbations of helical parameters. In contrast, disruptions caused by the GpC cross-link produced considerable conformational flexibility that precluded structure determination by NMR. AFM imaging of cross-link-containing plasmid DNA showed that the increased flexibility observed in the GpC cross-link persists when it is embedded into much larger DNA fragments. These differences may account for the different repair efficiencies seen in NER deficient cells.

Experimental demonstration of T:(G:G:G:G):T hexad and T:A:A:T tetrad alignments within a DNA quadruplex stem.

A template-based approach was used to design unprecedented architectural motifs into a known DNA framework. The structure formed by the sequence d(GCGGTTGGAT) in 0.1 M Na(+) solution has been determined using molecular dynamics simulations constrained by distance and dihedral restraints derived from NMR experiments. The molecular topology has been previously observed for the sequence d(GCGGTGGAT) (Webba da Silva, M. (2003) Biochemistry 42, 14356-65). Insertion of a single thymine into the double chain reversal formed by the segment GGTGG results in the unprecedented experimental demonstration of a T:(G:G:G:G):T hexad. The bi-stranded hexad results from the pairing alignment of two G(T-G) triads. Each triad results from recognition of the sheared edge of a guanine by the Watson-Crick edge of a thymine of the segment GGTTGG. The alignment is stabilized by base-stacking of the thymine to the sugar pucker of the preceding thymine. The latter is involved in formation of the T:A:A:T tetrad alignment by forming a hydrogen bond with the free amino proton of a Watson-Crick aligned A:A mispair. We have thus established that residues in double chain reversal loops linking juxtaposed tetrads of a quadruplex stem may facilitate formation of yet unknown hydrogen bond alignments. By employing a systematic approach analysis of sequence motifs appearing in double chain reversals, bridging tetrad layers should allow for the prediction of topologies and architectural motifs appearing in biologically relevant genomic regions.

Kinetics and thermodynamics of G-quadruplexes.

The melting of tetramolecular DNA or RNA quadruplexes is kinetically irreversible. However, rather than being a hindrance, this kinetic inertia allows us to study association and dissociation processes independently. General rules have been extended to longer DNA motifs or sequences containing modified bases such as 8-oxo or 7-deaza guanine. Results were compared with the canonical TG4T and TG5T tetramers: we demonstrate huge differences (up to 10(5)-fold) in the association constants of these quadruplexes depending on primary sequence.

Association of DNA quadruplexes through G:C:G:C tetrads. Solution structure of d(GCGGTGGAT).

The structure formed by the DNA sequence d(GCGGTGGAT) in a 100 mM Na(+) solution has been determined using molecular dynamics calculations constrained by distance and dihedral restraints derived from NMR experiments performed at isotopic natural abundance. The sequence folds into a dimer of dimers. Each symmetry-related half contains two parallel stranded G:G:G:G tetrads flanked by an A:A mismatch and by four-stranded G:C:G:C tetrads. Each of the two juxtaposed G:C:G:C tetrads is composed of alternating antiparallel strands from the two halves of the dimer. For each single strand, a thymine intersperses a double chain reversal connecting the juxtaposed G:G:G:G tetrads. This architecture has potential implications in genetic recombination. It suggests a pathway for oligomerization involving association of quadruplex entities through GpC steps.