Analysis of the effect of LRP-1 silencing on the invasive potential of cancer cells by nanomechanical probing and adhesion force measurements using atomic force microscopy.

Low-density lipoprotein receptor-related protein 1 (LRP-1) can internalize proteases involved in cancer progression and is thus considered a promising therapeutic target. However, it has been demonstrated that LRP-1 is also able to regulate the endocytosis of membrane-anchored proteins. Thus, strategies that target LRP-1 to modulate proteolysis could also affect adhesion and cytoskeleton dynamics. Here, we investigated the effect of LRP-1 silencing on parameters reflecting cancer cells' invasiveness by atomic force microscopy (AFM). The results show that LRP-1 silencing induces changes in the cells' adhesion behavior, particularly the dynamics of cell attachment. Clear alterations in morphology, such as more pronounced stress fibers and increased spreading, leading to increased area and circularity, were also observed. The determination of the cells' mechanical properties by AFM showed that these differences are correlated with an increase in Young's modulus. Moreover, the measurements show an overall decrease in cell motility and modifications of directional persistence. An overall increase in the adhesion force between the LRP-1-silenced cells and a gelatin-coated bead was also observed. Ultimately, our AFM-based force spectroscopy data, recorded using an antibody directed against the ß1 integrin subunit, provide evidence that LRP-1 silencing modifies the rupture force distribution. Together, our results show that techniques traditionally used for the investigation of cancer cells can be coupled with AFM to gain access to complementary phenotypic parameters that can help discriminate between specific phenotypes associated with different degrees of invasiveness.

Targeting focal adhesion assembly by ethoxyfagaronine prevents lymphoblastic cell adhesion to fibronectin.

BACKGROUND: Leukemic cell adhesion to proteins of the bone marrow microenvironment provides signals which control morphology, motility and cell survival. We described herein the ability of ethoxyfagaronine (etxfag), a soluble synthetic derivative of fagaronine, to prevent leukemic cell adhesion to fibronectin peptide (FN/V). METHODS: Phosphorylation of fak and pyk2 were evaluated by immunoblotting. Labelled proteins were localized by confocal microscopy. PI 3-kinase activity was evaluated by in vitro kinase assay. RESULTS: Subtoxic concentration of etxfag reduced L1210 cell adhesion to FN/V dependently of ß1 integrin engagement. Etxfag impaired FN-dependent formation of ß1 clustering without modifying ß1 expression at the cell membrane. This was accompanied by a decrease of focal adhesion number, a diminution of fak and pyk2 phosphorylation at Tyr-576, Tyr-861 and Tyr-579, respectively leading to their dissociations from ß1 integrin and inhibition of PI 3-kinase activity. Etxfag also induced a cell retraction accompanied by a redistribution of phosphorylated fak and pyk2 in the perinuclear region and lipid raft relocalization. CONCLUSION: Through its anti-adhesive potential, etxfag, combined with conventional cytotoxic drugs could be potentially designed as a new anti-leukemic drug.

Elastin-derived peptides enhance melanoma growth in vivo by upregulating the activation of Mcol-A (MMP-1) collagenase.

BACKGROUND: Elastin peptides possess several biological activities and in vitro data suggest they could be involved in the early phase of melanoma growth. METHODS: Using diverse in vitro and in vivo techniques (cell proliferation, invasion and migration assays, zymography, western blots, collagen degradation assay, reverse transcription PCR, melanoma allographs and immunohistochemistry), we analysed the effect of elastin-derived peptides (EDPs) on B16F1 melanoma growth and invasion, as well as on the proteolytic systems involved. RESULTS: We found that EDPs dramatically promote in vivo tumour development of B16F1 melanoma, as well as their in vitro migration and invasion. The inhibition of serine proteases and matrix metalloproteinases (MMPs) activities, by aprotinin and galardin, respectively, demonstrated that these enzymes were involved in these processes. However, we found that EDPs did not increase urokinase-type plasminogen activator, tissue-type plasminogen activator or MMP-2 expression and/or activation, neither in vitro nor in vivo. Nevertheless, we observed a strong increase of pro-MMP-9 secretion in EDPs-treated tumours and, more importantly, an increase in the expression and activation of the murine counterpart of MMP-1, named murine collagenase-A (Mcol-A). Moreover, we show that plasminogen system inhibition decreases collagen degradation by this enzyme. Finally, the use of a specific blocking antibody against Mcol-A abolished EDP-induced B16F1 invasion in vitro, showing that this MMP was directly involved in this process. CONCLUSION: Our data show that in vivo, EDPs are involved in melanoma growth and invasion and reinforced the concept of elastin fragmentation as a predictive factor.

Hydroxyethylstarch microcapsules: a preliminary study for tumor immunotherapy application.

The objective of this work was to prepare microcapsules which would allow protection and slow release of antigens used for melanoma immunotherapy treatment. Hydroxyethylstarch (HES) microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride (TC). They were characterized with respect to morphology (microscopy) and size (in the 4-15 microm range). Bovine serum albumin (BSA) was used as model protein for loading and release studies. Microcapsules were loaded with solutions at different protein concentrations (0.5-5%). The maximum loading efficiency (20%) was observed with the concentration of 2.5%, which allowed a loading capacity near 100%. Confocal laser scanning microscopy (CLSM) visualization showed that BSA was entrapped within the microcapsules and not only associated to their outer surface. BSA-release studies showed a 20% BSA release within 30 min while 80% remained entrapped in the microcapsules for 4 days. Microcapsules were degraded by alpha-amylase and addition of esterase to alpha-amylase enhanced slightly their degradation. In vitro studies on melanoma cells showed that HES microcapsules were non-toxic. Preliminary in vivo studies demonstrated that microcapsules were biodegradable after intraperitoneal injection (i.p.). The observation of peritoneal wash showed a complete degradation within 7 days, indicating a possible application as an in vivo drug delivery system especially to enhance the presentation of antigens.

Inhibition of multidrug resistance by immunisation with synthetic P-glycoprotein-derived peptides.

Overexpression of the membrane glycoprotein (P170) represents the most common multidrug resistance (MDR) mechanism in cancer therapy. Specific auto-antibodies to extracellular loops 1, 2 and 4 of murine P170 were elicited in mice using palmitoylated synthetic peptides reconstituted in liposomes, with or without Lipid A, and resuspended in alum. IgM antibodies were detected 14 days following the first injection and IgG1 became predominant after the third challenge. Animals did not show any auto-immune symptoms or induced toxicity up to 18 months after the immunisation. Previous immunisations of mice using liposomes with MDR1 peptides increases the efficacy of chemotherapy treatments with doxorubicin and vinblastine against P388 R cells with increase of 77% in the survival half time in the immunised group. Sera from the immunised mice were also effective in reducing cellular resistance to vinblastine and doxorubicin in vitro. Taken together, these data suggest that this immunisation approach might have potential clinical applications.

Synthesis, biological activity and comparative analysis of DNA binding affinities and human DNA topoisomerase I inhibitory activities of novel 12-alkoxy-benzo[c]phenanthridinium salts.

New antitumor 12-alkoxy-benzo[c]phenanthridinium derivatives were obtained in high yields through multistep syntheses. Analysis of DNA binding and human DNA topoisomerase I inhibitory activities demonstrates that new compounds, combining 2, 6, and 12 substitutions, interact strongly with DNA and exhibit important topoisomerase I inhibition. The cytotoxicities against solid tumor cell lines are also determined and compared with those for fagaronine and ethoxidine.

Human DNA topoisomerase I inhibitory activities of synthetic polyamines: relation to DNA aggregation.

DNA aggregation by polyamines has acquired importance as a prerequisite for the cellular uptake of DNA for gene therapy. Intracellular polyamines are constitutive components of mammalian cells and their availability is critical for cell proliferation. Interference of polyamine biosynthesis by synthetic polyamines leads to cytotoxicity. Optimization of the polyamine structural parameters is necessary to control their DNA aggregation, cytotoxic or enzyme inhibitory activities. We designed two series of tetra- and hexamines and compared their human DNA topoisomerase I (top1) inhibitory effects with the DNA aggregation properties. We show that hexamines are more efficient inhibitors of DNA relaxation by top1 than tetramines and that they suppress the top1-mediated DNA cleavage while tetramines do not. The DNA aggregation abilities within two series of polyamines correlate with the length of their central methylene chain. By contrast, the top1 inhibition within two series does not show the same correlation but demonstrates a threshold inhibitory effect on going from the (CH(2))(12) to the (CH(2))(14) central chain. We show further that the structures of DNA aggregates formed by polyamines with the (CH(2))(10-12) or with the (CH(2))(14-16) chains are very different. The first are a fluid cholesteric-type phases, whereas the second are well-structured aggregates similar to columnar liquid crystals with high packing density of DNA duplexes. The structures of polyamines-induced DNA aggregates are proposed to be crucial for top1 catalysis. The structure-function correlation described here may serve as a guide for rational design of polyamines with desired DNA-aggregation or anti-top1 activities.

Molecular determinants of site-specific inhibition of human DNA topoisomerase I by fagaronine and ethoxidine. Relation to DNA binding.

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016-50 microM concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(-1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH(2) group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.