New Symmetrically Esterified m-Bromobenzyl Non-Aminobisphosphonates Inhibited Breast Cancer Growth and Metastases

1 - ArticleBACKGROUND: Although there was growing evidence in the potential use of Bisphosphonates (BPs) in cancer therapy, their strong osseous affinities that contrast their poor soft tissue uptake limited their use. Here, we developed a new strategy to overcome BPs hydrophilicity by masking the phosphonic acid through organic protecting groups and introducing hydrophobic functions in the side chain. METHODOLOGY/PRINCIPAL FINDINGS: We synthesized non-nitrogen BPs (non N-BPs) containing bromobenzyl group (BP7033Br) in their side chain that were symmetrically esterified with hydrophobic 4-methoxphenyl (BP7033BrALK) and assessed their effects on breast cancer estrogen-responsive cells (T47D, MCF-7) as well as on non responsive ones (SKBR3, MDA-MB-231 and its highly metastatic derived D3H2LN subclone). BP7033Br ALK was more efficient in inhibiting tumor cell proliferation, migration and survival when compared to BP7033Br. Although both compounds inhibited tumor growth without side effects, only BP7033Br ALK abrogated tumor angiogenesis and D3H2LN cells-induced metastases formation. CONCLUSION/SIGNIFICANCE: Taken together these data suggest the potential therapeutic use of this new class of esterified Bisphosphonates (BPs) in the treatment of tumor progression and metastasis without toxic adverse effects

Invading Basement Membrane Matrix Is Sufficient for MDA-MB-231 Breast Cancer Cells to Develop a Stable In Vivo Metastatic Phenotype

1 - ArticleIntroduction: The poor efficacy of various anti-cancer treatments against metastatic cells has focused attention on the role of tumor microenvironment in cancer progression. To understand the contribution of the extracellular matrix (ECM) environment to this phenomenon, we isolated ECM surrogate invading cell populations from MDA-MB-231 breast cancer cells and studied their genotype and malignant phenotype. Methods: We isolated invasive subpopulations (INV) from non invasive populations (REF) using a 2D-Matrigel assay, a surrogate of basal membrane passage. INV and REF populations were investigated by microarray assay and for their capacities to adhere, invade and transmigrate in vitro, and to form metastases in nude mice. Results: REF and INV subpopulations were stable in culture and present different transcriptome profiles. INV cells were characterized by reduced expression of cell adhesion and cell-cell junction genes (44% of down regulated genes) and by a gain in expression of anti-apoptotic and pro-angiogenic gene sets. In line with this observation, in vitro INV cells showed reduced adhesion and increased motility through endothelial monolayers and fibronectin. When injected into the circulation, INV cells induced metastases formation, and reduced injected mice survival by up to 80% as compared to REF cells. In nude mice, INV xenografts grew rapidly inducing vessel formation and displaying resistance to apoptosis. Conclusion: Our findings reveal that the in vitro ECM microenvironment per se was sufficient to select for tumor cells with a stable metastatic phenotype in vivo characterized by loss of adhesion molecules expression and induction of proangiogenic and survival factors

Plastocyanin-cytochrome F Interactions: The Influence Of Hydrophobic Patch Mutations Studied By Nmr Spectroscopy

Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 ((2) 103 M-1 for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemicalshift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed

Mutagenesis of prochlorothrix plastocyanin reveals additional features in photosystem I interactions

5 pages, 4 figures.-- PMID: 12509429 [PubMed].-- Onlise version published Dec 30, 2002.Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.Research at Sevilla was supported by the Dirección General de Investigación Científica y Técnica (Grant BMC2000-0444), European Union (Networks ERB-FMRX-CT98-0218 and HPRN-CT1999-00095), and Junta de Andalucía (CVI-198). Work at Bowling Green was supported by National Science Foundation Grants MCB-9634049 and BIR-0070334.Peer reviewe

BP7033Br and BP7033Br ALK inhibited D3H2LN tumor growth and esterified analogue completely inhibited angiogenesis.

<p>(A) D3H2LN cells were inoculated in nude mice as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004685#s4" target="_blank">Materials and Methods</a>”. After 1 week, mice were treated with BPs (11 mg/kg), twice a week, for 21 days. Each column represents the mean of tumor volume (mm³) (±SD, n = 7). Body weight (BW) ratio was determined for each group (B). Endothelial cells in tumor sections were stained in controls (C), BP7033Br (D) and BP7033Br ALK (E) Microvessels were indicated by arrows and necrosis area by double asterisks (magnification ×100). Quantification of micro-vessel density (F). Each column represents a mean (±SD) of three independent experiments. *<i>P</i>_{BP7033Br and BP7033Br ALKversus control}<0.05.</p

BP7033Br and BP7033Br ALK inhibited MDA-MB-231 breast cancer cell migration, invasion, MMP-9 and MMP-2 activities.

<p>BPs inhibited MDA-MB-231 breast cancer cell migration (A) and invasion (B). Cells (2.5×10⁵) with 125 µM of BPs were added to each 8 µm-insert in the upper chamber of boyden chamber. After 24 h, cells invading the chamber were fixed, stained and counted as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004685#s4" target="_blank">Materials and Methods</a>”. BPs inhibited MMP-9 and MMP-2 activities (C and D, respectively). Lyophilized conditioned media were normalized to the number of cells and subjected to 10% SDS-polyacrylamide gels containing 1 mg/mL gelatine. Lane 1, 2 and 3 represent the control, BP7033Br and BP7033Br ALK conditioned medium of treated cells, respectively. Each column represents a mean (±SD) of three independent experiments. *<i>P</i>_{versus MDA-MB-231 control}<0.05, ** <i>P</i>_{versus D3H2LN control}<0.05.</p

BP7033Br and BP7033Br ALK inhibited MDA-MB-231 breast cancer cell cycle progression.

<p>Distribution of MDA-MB-231 (A) and D3H2LN (B) cells treated with BPs in different cell cycle phases was determined as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004685#s4" target="_blank">Materials and Methods</a>”. Histograms show the percentage of MDA-MB-231 (C) and D3H2LN (D) cell repartition. Each column represents a mean (±SD) of three independent experiments. *P _{control versus BP7033BrALK} and ** <i>P </i>_{control versus BP7033Br}<0.05.</p