Permanent Occlusion of Feeding Arteries and Draining Veins in Solid Mouse Tumors by Vascular Targeted Photodynamic Therapy (VTP) with Tookad

Background

Antiangiogenic and anti-vascular therapies present intriguing alternatives to cancer therapy. However, despite promising preclinical results and significant delays in tumor progression, none have demonstrated long-term curative features to date. Here, we show that a single treatment session of Tookad-based vascular targeted photodynamic therapy (VTP) promotes permanent arrest of tumor blood supply by rapid occlusion of the tumor feeding arteries (FA) and draining veins (DV), leading to tumor necrosis and eradication within 24–48 h.

Methodology/Principal Findings

A mouse earlobe MADB106 tumor model was subjected to Tookad-VTP and monitored by three complementary, non-invasive online imaging techniques: Fluorescent intravital microscopy, Dynamic Light Scattering Imaging and photosensitized MRI. Tookad-VTP led to prompt tumor FA vasodilatation (a mean volume increase of 70%) with a transient increase (60%) in blood-flow rate. Rapid vasoconstriction, simultaneous blood clotting, vessel permeabilization and a sharp decline in the flow rates then followed, culminating in FA occlusion at 63.2 sec±1.5SEM. This blockage was deemed irreversible after 10 minutes of VTP treatment. A decrease in DV blood flow was demonstrated, with a slight lag from FA response, accompanied by frequent changes in flow direction before reaching a complete standstill. In contrast, neighboring, healthy tissue vessels of similar sizes remained intact and functional after Tookad-VTP.

Conclusion/Significance

Tookad-VTP selectively targets the tumor feeding and draining vessels. To the best of our knowledge, this is the first mono-therapeutic modality that primarily aims at the larger tumor vessels and leads to high cure rates, both in the preclinical and clinical arenas.     

Synthesis of twisted intercalating nucleic acids possessing acridine derivatives. Thermal stability studies

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been synthesized via the postsynthetic modifications of oligonucleotides possessing insertions of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) at the 5'-end or in the middle as a bulge. In the first postsynthetic step, oligonucleotides 8 and 9 on the CPG support were treated with a Sonogashira coupling reaction mixture containing 9-chloro-2-ethynylacridine or 9-chloro-2-iodoacridine, respectively. After the postsynthetic step, treatment of the oligonucleotides with 32% aq ammonia or 50% ethanolic solution of tris(2-aminoethyl)amine led to the substitution of chloride on acridine concurrent with deprotection of the bases and cleavage of the oligonucleotides from CPG. Molecular modeling of the parallel triplex with a bulged insertion of the monomer (R)-3-O-[4-(9-aminoacridin-2-ylethynyl)benzyl]glycerol in the triplex-forming oligonucleotide (TFO) showed that the acridine moiety was stacking between the bases of the duplex, while phenyl was placed between the bases of the TFO. Thermal denaturation studies and fluorescence properties of TINA-acridine oligonucleotide duplexes and triplexes are discussed.     

Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H(2)O(2) might activate Axl by promoting autophosphorylation. H(2)O(2) rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H(2)O(2) was concentration-dependent with EC(50) of approximately 500 microm. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H(2)O(2)-induced Axl phosphorylation by 50%. Axl phosphorylation by H(2)O(2) was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H(2)O(2), and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H(2)O(2)in Axl(+/+) mouse aorta but significantly inhibited in Axl(-/-) aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl(-/-) mice compared with Axl(+/+) mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.     

Action of hypoxia on different types of calcium channels in hippocampal neurons

Whole-cell patch clamp and polarographic oxygen partial pressure (pO2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO2 to 15-30 mm Hg induced a potentiation of HVA Ca2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.     

On the conformation of the COOH-terminal domain of the large mechanosensitive channel MscL

COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear. The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. 1998. SCIENCE: 282:2220-2226). The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions. Based on the structure of pentameric cartilage protein, we modeled the COOH-terminal region of E. coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle. Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments. The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant. The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model. Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating. Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Delta110-136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially. The Delta110-136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls. The data strongly suggest that in contrast to previous gating models (Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. 2001a. NATURE: 409:720-724.), S3 domains are stably associated in both closed and open conformations. The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.     

A photosystem II-associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution

We show for the first time that Cah3, a carbonic anhydrase associated with the photosystem II (PSII) donor side in Chlamydomonas reinhardtii, regulates the water oxidation reaction. The mutant cia3, lacking Cah3 activity, has an impaired water splitting capacity, as shown for intact cells, thylakoids and PSII particles. To compensate this impairment, the mutant overproduces PSII reaction centres (1.6 times more than wild type). We present compelling evidence that the mutant has an average of two manganese atoms per PSII reaction centre. When bicarbonate is added to mutant thylakoids or PSII particles, the O2 evolution rates exceed those of the wild type by up to 50%. The donor side of PSII in the mutant also exhibits a much higher sensitivity to overexcitation than that of the wild type. We therefore conclude that Cah3 activity is necessary to stabilize the manganese cluster and maintain the water-oxidizing complex in a functionally active state. The possibility that two manganese atoms are enough for water oxidation if bicarbonate ions are available is discussed.     

Ribosomal protein S1 from Thermus thermophilus: its detection,identification and overproduction

Ribosomal protein S1 has been identified in Thermus thermophilus ribosomes. The gene of ribosomal protein S1 from Thermus thermophilus has been cloned and overexpressed in Escherichia coli. A procedure for purification of the protein has been developed.     

Barium acylpyrazolonate derivatives stabilized by O- and N-donor ligands: synthesis, spectral and structural characterization

New Ba acylpyrazolonate derivatives of formula [Ba(Q)₂(tetraglyme)] (HQ = HQ^tbu = 1-Ph-3-Me-4-C(O)CH₂Bu^t-5-pyrazolone; tetraglyme = 2,5,8,11,14-pentaoxapentadecane), [Ba(Q)₂(tmeda)₂] (HQ = HQ^tbu or HQ^piv, where HQ^piv = 1-Ph-3-Me-4-C(O)Bu^t-5-pyrazolone; tmeda = N,N,N′,N′-tetramethylethylenediamine), [Ba(Q)₂(pmdien)(H₂O)] (HQ = HQ^tbu or HQ^piv; pmdien = N,N,N′,N″,N″-pentamethyldiethylenetriamine) and [Ba(Q)₂(pmdien)(Meim)] (HQ = HQ^tbu or HQ^piv; Meim = 1-methylimidazole) have been synthesized and analytically and spectroscopically characterized. They are mononuclear air and solution stable compounds containing an eight-coordinated barium atom. The X-ray crystal structures of the hydrates [Ba(Q^tbu)₂(pmdien)(H₂O)] and [Ba(Q^piv)₂(pmdien)(H₂O)] show the water molecule directly bonded to Ba and involved in intermolecular H-bonding network. In the X-ray crystal structure of [Ba(Q^piv)₂(pmdien)(Meim)], the Meim ligand substitutes the water of previous derivatives and decreases the strength of intermolecular interactions, lowering the melting point. The derivative [Ba(Q^tbu)₂(pmdien)(NEt₃)] has been prepared from interaction of [Ba(Q^tbu)₂(pmdien)(H₂O)] with excess triethylamine in acetonitrile.     

Cellular energization protects the photosynthetic machinery against salt-induced inactivation in Synechococcus

The effects of the energization of cells by light and by exogenous glucose on the salt-induced inactivation of the photosynthetic machinery were investigated in the cyanobacterium Synechococcus sp. PCC 7942. The incubation of the cyanobacterial cells in a medium supplemented with 0.5 M NaCl induced a rapid decline with a subsequent slow decline, in the oxygen-evolving activity of Photosystem (PS) II and in the electron-transport activity of PSI. Light and exogenous glucose each protected PSII and PSI against the second phase of the NaCl-induced inactivation. The protective effects of light and glucose were eliminated by an uncoupler of phosphorylation and by lincomycin, an inhibitor of protein synthesis. Light and glucose had similar effects on the NaCl-induced inactivation of Na(+)/H(+) antiporters. After photosynthetic and Na(+)/H(+)-antiport activities had been eliminated by the exposure of cells to 0.5 M NaCl in the darkness, both activities were partially restored by light or exogenous glucose. This recovery was prevented by lincomycin. These observations suggest that cellular energization by either photosynthesis or respiration, which is necessary for protein synthesis, is important for the recovery of the photosynthetic machinery and Na(+)/H(+) antiporters from inactivation by a high level of NaCl.