Development of peptide-based conjugates for targeted tumour therapy is a current research topic providing new possibilities in cancer treatment. In this study, VHLGYAT heptapeptide selected by phage display technique for HT-29 human colon cancer was investigated as homing peptide for drug delivery. Daunomycin was conjugated to the N-terminus of the peptide directly or through Cathepsin B cleavable spacers. Conjugates showed moderate in vitro cytostatic effect. Therefore, sequence modifications were performed by Ala-scan and positional scanning resulting in conjugates with much higher bioactivity. Conjugates in which Gly was replaced by amino acids with bulky apolaric side chains provided the best efficacy. The influence of the cellular uptake, stability and drug release on the anti-tumour activity was investigated. It was found that mainly the difference in the cellular uptake of the conjugates generated the distinct effect on cell viability. One of the most efficient conjugate Dau=Aoa-LRRY-VHLFYAT-NH 2 showed tumour growth inhibition on orthotopically developed HT-29 colon cancer in mice with negligible toxic side effect compared to the free drug. We also indicate that this sequence is not specific to HT-29 cells, but it has a remarkable effect on many other cancer cells. Nevertheless, the Phe-containing conjugate was more active in all cases compared to the conjugate with the parent sequence. The literature data suggested that this sequence is highly overlapped with peptides that recognize Hsp70 membrane bound protein overexpressed in many types of tumours.

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Recently we have characterized the DNA and nucleoprotein (NP) binding of bis(4-Nmethylpyridyl)-15,20-di(4-carboxyphenyl)porphyrin (BMPCP) and meso-tri(4-Nmethylpyridyl)-mono(4-carboxyphenyl)porphyrin (TMPCP) and their tetra-peptide conjugates (BMPCP-4P 2 and TMPCP-4P, respectively). In this work we investigated the interaction of TMPCP conjugated to the tetra-peptide branches of branched chain polymeric polypeptide with poly-L-lysine backbone (AK) with DNA or NP using spectroscopic methods.Analysis of absorption spectra revealed the external binding, but no intercalation of TMPCP-AK to DNA. There was no evidence for the interaction between TMPCP-AK and encapsidated DNA. Furthermore, we examined the cellular uptake of BMPCP and TMPCP and their tetra-or polypeptide conjugates by flow cytometry and analyzed how charge, size and structure of the compounds affect their incorporation. In comparison, liposomal association constants of these derivatives were determined. BMPCP-4P 2 accumulated the most, and porphyrins with two positive charges (BMPCP and BMPCP-4P 2 ) showed better accumulation than the tri-cationic TMPCP or TMPCP-4P.Cellular uptake of polycationic TMPCP-AK was significantly lower than that of the free or tetra-peptide conjugated derivatives. The sub-cellular localization of all the five compounds was investigated in co-localization studies by confocal microscopy with special attention to their nuclear localization. Neither free nor conjugated BMPCP or TMPCP were co-localized with nuclear marker. Instead, these derivatives showed co-localization with lysosomal and mitochondrial fluorescent probes. TMPCP-AK conjugate had different localization pattern appearing mainly in mitochondria and cytoplasmic vesicles.Our results may contribute to the further design of DNA targeting porphyrin based constructs.

Cationic macromolecular carriers can be effective carriers for small molecular compounds, drugs, epitopes or nucleic acids. Polylysine based polymeric branched polypeptides have been systematically studied on the level of cells and organisms as well. In the present study we report on our findings on the cellular uptake characteristics of nine structurally related polylysine based

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Colorectal cancer (CRC) has a high incidence and is one of the leading causes of cancer-related death. The accumulation of cancer-associated fibroblasts (CAF) induces an aggressive, stem-like phenotype in tumor cells, and it indicates a poor prognosis. However, cellular heterogeneity among CAFs and the targeting of both stromal and CRC cells are not yet well resolved. Here, we identified CD142high fibroblasts with a higher stimulating effect on CRC cell proliferation via secreting more hepatocyte growth factor (HGF) compared to CD142low CAFs. We also found that combinations of inhibitors that had either a promising effect in other cancer types or are more active in CRC compared to normal colonic epithelium acted synergistically in CRC cells. Importantly, heat shock protein 90 (HSP90) inhibitor selected against CD142high fibroblasts, and both CRC cells and CAFs were sensitive to a BCL-xL inhibitor. However, targeting mitogen-activated protein kinase kinase (MEK) was ineffective in fibroblasts, and an epigenetic inhibitor selected for a tumor cell population with markers of aggressive behavior. Thus, we suggest BCL-xL and HSP90 inhibitors to eliminate cancer cells and decrease the tumor-promoting CD142high CAF population. This may be the basis of a strategy to target both CRC cells and stromal fibroblasts, resulting in the inhibition of tumor relapse.

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The DNA sequence preferences of nucleosomes can be captured in a probabilistic model that ascribes a probability or, equivalently, an energy to any 147-bp sequence. This is generally done using a Markov chain model, where the total probability of a sequence is calculated from the probability distributions of subsequences of some length (usually dinucleotides) under the assumption that long-distance correlations are less important. These probability distributions must be trained on some subset of the entire sequence space, which has in the past been done experimentally, with certain statistical limitations and caveats. Using a novel simulation method, we are now able to produce large ensembles for training in silico, derived from an underlying energetic nucleosome model. This allows us for the first time to quantitatively examine how well such short-range probabilistic models approximate reality. In a next step, a probabilistic model trained on a quality sequence ensemble can then be used to predict energy landscapes for DNA sequences with negligible computational cost. This will allow us to test the idea that DNA may have evolved mechanical signals for nucleosome positioning, by simulating such evolution and comparing the patterns we find to patterns found in real genomes. 1995-Pos Board B139Continuously Scanning DNA with Nanopore MspA Nanopore sequencing is a promising next-generation technology that is being enabled by the biological nanopore MspA. In this sequencing technique, current is driven through the~1 nm constriction of MspA. Single stranded DNA molecules are first drawn into the pore by an applied voltage. Next, a polymerase or helicase moves the DNA by discrete steps as it interacts with the pore. As the DNA moves through the pore, different nucleobases modulate the pore's conductance to varying extents, allowing for extraction of sequence information from the current trace. In this experiment, we gain more complete information by using a variable applied voltage to stretch the DNA within the pore. Changing the voltage continuously repositions the DNA in the constriction. Coupling this voltage-induced movement with the enzyme-induced motion we reconstruct a profile characteristic of the DNA as it is pulled continuously through the constriction. This profile provides a tool for improving the de novo sequencing accuracy of the nanopore technique. of. Higher order chromatin structures and nuclear genome organization play a key role in regulating gene functions in addition to other epigenetic markers such as DNA and histone modifications, which have been extensively studied. Genetic variations in the human genome are known to lead to several human diseases but little is known yet about how the structural organization of the chromatin fibers affects gene regulation, cell differentiation, and disease pathways. Understanding the dynamics of native chromatin in living cells by imaging and tracing specific gene loci and nuclear transcripts would allow us to uncover the correlation between chromosome organizati...