Anti-invasion and anti-metastasis effects of Dandelion (Taraxacum officinale) hydroalcoholic extract on glioblastoma multiforme cell line model

OBJECTIVE: Glioblastoma multiforme is one of the malignant brain tumors and despite recent advancements in cancer treatment remains largely incurable. Cancer invasion has a cascade of interrelated and sequential steps, including cell adhesion, extracellular matrix degradation, and cell movement. Hence, inhibition of the invasion-associated steps could be a potential strategy for prolonging the life of patients. This study aimed to evaluate the anti-invasion and anti-metastasis effects of Dandelion (Taraxacum officinale) hydro-alcoholic extract on glioblastoma multiforme cell line (U87MG). MATERIALS AND METHODS: The hydro-alcoholic extract was prepared, and the cell line was treated with 1000, 500, 250, 125, and 62.5 µg / ml of extract for 24, 48, and 72 hr. Cell viability was evaluated. The effect of extract (IC50 concentration) on cancer cell invasion potential was tested. The expression levels of MMP-2, MMP-9, TIMP-1, TIMP-2, uPA, uPAR, p38MAPK, ERK1/2, and SAPK/JNK were analyzed. Comparisons between groups were performed by Tukey’s test one-way analysis of variance and differences were considered significant when p < 0.05. RESULTS: After treatment with extract, the cells viability was decrease in a concentration- and time-dependent. IC50 concentration of dandelion extract significantly decreased the cell migration by 32% (p<0.05), cell invasion potential by 77% (p<0.05) and cell adhesion by 51% (p<0.05). Also, the expression levels of proteolytic enzymes associated with matrix and base membrane degradation (MMP-2, MMP-9, and uPA) were decreased and the levels of their endogenous inhibitors (TIMP1 and TIMP2) were increased. Moreover, the p38MAPK and SAPK/JNK signaling pathway, which stimulates proteolytic enzymes and matrix degradation, was inhibited by extract treatment. CONCLUSIONS: Dandelion extract reduced the viability and invasion potential of the glioblastoma cells by regulating proteolytic enzymes and matrix dynamics through the p38MAPK and SAPK/JNK pathway

Oncogenic role of connective tissue growth factor is associated with canonical TGF-β cascade in colorectal cancer

TGF-β signaling pathways promote tumour development and control several downstream genes such as CTGF and MMPs. This study aimed to investigate the association between CTGF and MMP-1 mRNA expressions with clinicopathological status and survival rate in colorectal cancer patients. We investigated expression levels of CTGF and MMP-1 genes in paraffin-embedded tumours and adjacent normal tissue blocks (ADJ) by Real Time-PCR. Then, the expression of Smad2 and Smad4 proteins in the TGF-β canonical pathway was evaluated by immunohistochemistry. Finally, the correlation between CTGF, MMP-1, and the canonical TGF-β-signalling pathway with the clinicopathological features was investigated. Expression levels of MMP-1and CTGF were higher in tumours compared with adjacent normal tissues. Overexpression levels of MMP-1 and CTGF were associated with lymph node metastasis, distant metastasis, tumour histopathological grading, advanced stage, and poor survival (p 0.05). Additionally, a significant association between the upregulation of MMP-1 and tumour location was noted. Upregulation of Smad2 and Smad4 proteins were also significantly correlated with lymph node metastasis, distant metastasis, advanced stage, and poor survival (p 0.0001). This study showed that canonical TGF-β signalling regulates both CTGF and MMP-1 expression and CRC progression. Moreover, TGF-β signalling and its downstream genes could be used as novel biomarkers and novel approaches for targeted therapy in CRC

Designer Magnetoplasmonics with Nickel Nanoferromagnets

We introduce a new perspective on magnetoplasmonics in nickel nanoferromagnets by exploiting the phase tunability of the optical polarizability due to localized surface plasmons and simultaneous magneto-optical activity. We demonstrate how the concerted action of nanoplasmonics and magnetization can manipulate the sign of rotation of the reflected light’s polarization (i.e., to produce Kerr rotation reversal) in ferromagnetic nanomaterials and, further, how this effect can be dynamically controlled and employed to devise conceptually new schemes for biochemosensing. © 2011 American Chemical Society.A.D. and Z.P. acknowledge support from the Swedish Research Council and Swedish Foundation for Strategic Research (Framework program Functional Electromagnetic Metamaterials, project RMA08). J.Å. acknowledges support from the Swedish Research Council, the Swedish Foundation for Strategic Research (Future Research Leader Programme), and the G€oran Gustafsson Foundation. J.Å. is a Royal Swedish Academy of Sciences Research Fellow supported by a grant from the Knut and Alice Wallenberg Foundation. V.B. acknowledges the G€oran Gustafsson Foundation and the Blanceflor Boncompagni-Ludovisi Foundation. P.V. acknowledges funding from the Basque Government through the ETORGAI Program, Project No. ER- 2010/00032 and Program No. PI2009-17, the Spanish Ministry of Science and Education under Projects No. CSD2006-53 and No. MAT2009-07980. J.N. acknowledges funding for the Generalitat de Catalunya and the Spanish Ministry of Science and Education through No. 2009-SGR-1292 and No. MAT2010-20616-C02 projects.Peer Reviewe

Design and Evolution of Trans-Splicing Group I Intron Ribozymes

Group I introns are catalytic RNAs (ribozymes) capable of catalyzing their self-excision from precursor RNAs through two consecutive transesterification reactions. Although the ribozyme has evolved to perform this cis-splicing reaction in nature, man-made modifications to the 5' end of the ribozyme have allowed it to catalyze a trans- splicing reaction, in which it is able to replace the 3' portion of a substrate RNA with it's own 3' tail. The trans-splicing group I introns used in this thesis were variants of the Tth.L 1925 IC1 group I intron ribozyme found in Tetrahymena thermophila. The work contained in this dissertation aims to both utilize trans-splicing group I introns to further understand principles of RNA evolution, as well as develop and optimize a new trans- splicing variant of the ribozyme for future use in therapy. In the first study of this dissertation, a trans- splicing group I intron was used as a model system to examine the effect of selection pressure and recombination on evolving populations of RNA in a cellular environment. Four parallel evolutions were completed, two employing a low selection pressure, and two employing a high selection pressure. Ribozyme populations with higher efficiency, measured by cellular growth conferred by the ribozyme, resulted from evolutions performed at a low selection pressure. It was found that this increase in fitness was the result of a set of four mutations acting cooperatively. Fitness profiles of evolutionary intermediates revealed that a low selection pressure can increase the accessibility of evolutionary paths leading to the evolution of cooperative mutations. This finding not only adds to the understanding of natural RNA evolution, but also aids in the design of more efficient evolutions of RNA species. In the second study of this dissertation, a new trans-splicing variant of the group I intron was developed, capable of catalyzing the removal of internal sequences from pre-mRNA and joining the two flanking sequences, thereby generating a functional RNA. This group I intron has been termed the ̀spliceozyme' because its action is analogous to that of the spliceosome. The action performed by the spliceozyme give this system the ability to repair certain types of diseases caused by mis-splicing and therefore, the potential to be used therapeutically. To increase the efficiency and therapeutic potential of this system, the spliceozyme was evolved in E. coli cells, challenging it to more efficiently catalyze the removal of internal sequences. The most efficient variant contained a set of mutations resulting in increased product formation and decreased side product formation. This observed effect was seen in vitro, suggesting that this effect may increase spliceozyme efficiency in a range cell types. Future work will move this system into mammalian cells and optimize the spliceozyme for use in a mammalian system, thus developing it as a therapeutic too

Low selection pressure aids the evolution of cooperative ribozyme mutations in cells.

Understanding the evolution of functional RNA molecules is important for our molecular understanding of biology. Here we tested experimentally how two evolutionary parameters, selection pressure and recombination, influenced the evolution of an evolving RNA population. This was done using four parallel evolution experiments that employed low or gradually increasing selection pressure, and recombination events either at the end or dispersed throughout the evolution. As model system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 rounds of selection and amplification, including mutagenesis and recombination. The low selection pressure resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombination did not have a strong effect. Five mutations were responsible for the highest efficiency. The first mutation swept quickly through all four evolving populations, whereas the remaining four mutations accumulated later and more efficiently under low selection pressure. To determine why low selection pressure aided this evolution, all evolutionary intermediates between the wild type and the 5-mutation variant were constructed, and their activities at three different selection pressures were determined. The resulting fitness profiles showed a high cooperativity among the four late mutations, which can explain why high selection pressure led to inefficient evolution. These results show experimentally how low selection pressure can benefit the evolution of cooperative mutations in functional RNAs

Increased efficiency of evolved group I intron spliceozymes by decreased side product formation.

The group I intron ribozyme from Tetrahymena was recently reengineered into a trans-splicing variant that is able to remove 100-nt introns from pre-mRNA, analogous to the spliceosome. These spliceozymes were improved in this study by 10 rounds of evolution in Escherichia coli cells. One clone with increased activity in E. coli cells was analyzed in detail. Three of its 10 necessary mutations extended the substrate binding duplexes, which led to increased product formation and reduced cleavage at the 5'-splice site. One mutation in the conserved core of the spliceozyme led to a further reduction of cleavage at the 5'-splice site but an increase in cleavage side products at the 3'-splice site. The latter was partially reduced by six additional mutations. Together, the mutations increased product formation while reducing activity at the 5'-splice site and increasing activity at the 3'-splice site. These results show the adaptation of a ribozyme that evolved in nature for cis-splicing to trans-splicing, and they highlight the interdependent function of nucleotides within group I intron ribozymes. Implications for the possible use of spliceozymes as tools in research and therapy, and as a model for the evolution of the spliceosome, are discussed