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Background Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. Methods We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. Results We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. Conclusions Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. General significance Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) as gefitinib are standard treatment of non-small cell lung cancer (NSCLC), but resistance often occurs. This study demonstrates that NSCLC cells resistant to gefitinib (GR cells) displayed a significantly higher microsomal prostaglandin E synthase-1 (mPGES-1) expression and activity than parental cells. Overexpression of mPGES-1/prostaglandin E-2 (PGE-2) signaling in GR cells was associated with acquisition of mesenchymal and stem-like cell properties, nuclear EGFR translocation and tolerance to cisplatin. mPGES-1 inhibition reduced mesenchymal and stem-like properties, and nuclear EGFR translocation in GR cells. Consistently, inhibition of mPGES-1 activity enhanced sensitivity to cisplatin and responsiveness to gefitinib in GR cells. We propose the mPGES-1/PGE-2 signaling as a potential target for treating aggressive and resistant lung cancers.

Synthesis and biological evaluation of a new class of 1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine derivatives are reported. A preliminary cellular assay system using the tumor cell line A431 responding to epidermal growth factor (EGF) for its growth, shows that the new compounds are potent inhibitors of cell growth. The inhibition of tumor cell proliferation is not associated with blockage of EGF receptor (EGFR), but substantially due to the interference with the signalling pathway at the level of Src tyrosine kinase and at the level of the downstream effector signal mitogen activated protein kinases (MAPKs), ERK1-2.

We found that thrombospondin-1 (TSP-1) has opposite functions on angiogenesis depending on the nature of the proteolytic fragment released in vivo by the action of proteases. We studied the effect of the 25 and 140 kDa fragments of TSP-1 generated by its proteolytic cleavage on the cascade of mitogen activated protein kinase (MAPK) activation and matrix-metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) function and expression in microvascular endothelium. Post-capillary endothelial cells (CVEC) isolated from bovine heart were used. The 25 kDa fragment enhanced the upregulation of MMP-2 and -9 and reduced TIMP-2 expression leading to CVEC chemoinvasion. Conversely, the 140 kDa fragment blocked MMP-2 and -9 stimulation and doubled TIMP-2 expression, leading to inhibition of endothelial chemoinvasion induced by fibroblast growth factor-2 (FGF-2). MAPK activity (ERK1-2) was induced by TSP-1 and by the 25 kDa fragment, but not by the 140 kDa fragment which, however, promoted MAPK p38 activation. This evidence indicates that fragments originating from TSP-1 switch the pro- or anti-angiogenic phenotype in endothelium by targeting MAPK cascades with opposite functions on MMP/TIMP balance.

With the aim of improving the compatibility of biomaterials to be used for the construction of cardiovascular prosthesis, we have designed bioactive macromolecules resulting from chemical modifications of hyaluronic acid (Hyal). The stability constants of Cu(II) and Zn(II) complexes with the sulphated derivative of hyaluronic acid (HyalS 3.5 ) were evaluated. Two different complexes have been found for each metal ion, CuL, Cu(OH) 2 L and ZnL, Zn(OH) 2 L (L means the disaccharide unit of the ligands) in aqueous solution at 37°C. The dihydroxo Cu(II) complex was present in high percentage at pH=7.4. On the contrary, the Zn(II) ion was present with a relatively low percentage of both complexes. The ability to stimulate endothelial cell adhesion and migration was evaluated for Hyal, HyalS 3.5 and their complexes with Cu(II) and Zn(II) ions. The results revealed that Hyal and [Cu(OH) 2 HyalS 3.5 ] (4.5)− induced cell adhesion, while [ZnHyalS 3.5 ] (2.5)− and [Zn(OH) 2 HyalS 3.5 ] (4.5)− inhibited the process. The chemotactic activity of increasing concentrations of the above complexes was also evaluated, demonstrating that [Cu(OH) 2 HyalS 3.5 ] (4.5)− complex at 1 μM concentration was the most active in inducing cell migration. These results have been also strengthened by analysing adherent cell migration in agarose. In conclusion, sulphated hyaluronic acid coordinated to Cu(II) seems to be a promising matrix molecule for the construction of cardiovascular prosthesis.

Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, Bergamo, Italy National Institute for Cancer Research, Genoa, Italy Institute for Cancer Research and Treatment, University of Turin, Italy European Institute of Oncology, Milan, Italy Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy University of Siena, Italy European School of Oncology, Milan, Italy

We recently demonstrated that nitric oxide (NO) significantly contributes to the mitogenic effect of vascular endothelial growth factor (VEGF), suggesting a role for the NO pathway in the signaling cascade following kinase-derivative receptor activation in vascular endothelium. The aim of this study was to investigate the intracellular pathways linked to VEGF/NO-induced endothelial cell proliferation. We assessed the activity of the mitogen-activated protein kinase (MAPK) that is specifically activated by growth factors, extracellular-regulated kinase (ERK½), on cultured microvascular endothelium isolated from coronary postcapillary venules. ERK½ was immunoprecipitated, and its activity was assessed with an immunocomplex kinase assay. In endothelial cells exposed for 5 min to the NO donor drug sodium nitroprusside at a concentration of 100 μm, ERK½ activity significantly increased. VEGF produced a time- and concentration-dependent activation of ERK½. Maximal activity was obtained after 5 min of stimulation at a concentration of 10 ng/ml. The specific MAPK kinase inhibitor PD 98059 abolished ERK½ activation and endothelial cell proliferation in a concentration-dependent manner in response to VEGF and sodium nitroprusside. The NO synthase inhibitorN ω-monomethyl-l-arginine, as well as the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked the activation of ERK½ induced by VEGF, suggesting that NO and cGMP contributed to the VEGF-dependent ERK½ activation. These results demonstrate for the first time that kinase-derivative receptor activation triggers the NO synthase/guanylate cyclase pathway to activate the MAPK cascade and substantiates the hypothesis that the activation of ERK½ is necessary for VEGF-induced endothelial cell proliferation.

The nitric oxide (NO)-generating drug sodium nitroprusside (SNP) has been assayed on DNA synthesis in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). DNA synthesis was measured by [3H]-thymidine incorporation following 24 h exposure to SNP (0.1-100 microM). Cyclic GMP levels were also measured following drug exposure. SNP induced a dose-dependent increase in DNA synthesis of CVEC. Maximal effect was observed at 10 microM concentration (44% increment over basal condition). Methylene blue treatment reduced the effect of SNP on thymidine incorporation by 35.4%. After 5 min exposure to SNP, cyclic GMP levels increased up to 3 fold compared to basal. Treatment with N omega-monomethyl-L-arginine (L-NMMA) reduced levels of cyclic GMP to half basal, but did not modify the effect of SNP. Our results show that endogenous and exogenous NO production can modulate proliferation of endothelial cells at microvascular level.

We have investigated the possible effect of substance P (SP), a main mediator of neurogenic inflammation, on the growth of capillary vessels in vivo, and on the proliferation of cultured endothelial cells in vitro. Slow release preparations of SP were implanted into the avascular cornea of New Zealand White rabbits and vessel growth was monitored daily through a slit lamp stereomicroscope. SP (1–5 μg/pellet) induced a marked neovascularization. A selective NK-1 receptor agonist [β-Ala4, Sar9, Met(O2)11]-SP(4-11) also induced neovascularization. The addition of SP to serum-free cultured endothelial cells, isolated from bovine adrenals (BACE) and from human umbilical cord veins (HUVE), increased proliferation of both cell lines in a concentration-dependent manner with maximal activity at 10−8 M (BACE) and 10−10 M (HUVE). The selective NK-1 receptor agonist induced a similar proliferative action on both cell lines, while the selective NK-2 receptor agonist [β-Ala8]-NKA(4-10) and the selective NK-3 receptor agonist [MePhe7]-NKB had no significant effect. Two different SP antagonists [ d -Pro2, d -Trp7,9]-SP and [ d -Pro4, d -Trp7,9, Phe11]-SP (4-11) blocked the response to SP. These findings indicate that SP can directly stimulate the process of neovascularization, probably through induction of endothelial cell proliferation. This hitherto unraveled activity of SP could play a key role in the trophic action produced by activation of the efferent function of peripheral endings of primary sensory neurons.

On the basis of {sup 125}I-labeled plasminogen activator binding analysis the authors have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a K{sub d} of 0.8958{times}10{sup {minus}12} M. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.