Triggering apoptosis in cancer cells with an analogue of cribrostatin 6 that elevates intracellular ROS.

Elevation of reactive oxygen species (ROS) is both a consequence and driver of the upregulated metabolism and proliferation of transformed cells. The resulting increase in oxidative stress is postulated to saturate the cellular antioxidant machinery, leaving cancer cells susceptible to agents that further elevate their intracellular oxidative stress. Several small molecules, including the marine natural product cribrostatin 6, have been demonstrated to trigger apoptosis in cancer cells by increasing intracellular ROS. Here, we report the modular synthesis of a series of cribrostatin 6 derivatives, and assessment of their activity in a number of cell lines. We establish that placing a phenyl ring on carbon 8 of cribrostatin 6 leads to increased potency, and observe a window of selectivity towards cancer cells. The mechanism of activity of this more potent analogue is assessed and demonstrated to induce apoptosis in cancer cells by increasing ROS. Our results demonstrate the potential for targeting tumors with molecules that enhance intracellular oxidative stress.

HIF-1 promotes the expression of its α-subunit via an epigenetically regulated transactivation loop.

Hypoxia inducible factor-1 (HIF-1) directs the cellular response to low oxygen and plays a key role in tumour survival and growth. Here we use an inhibitor of the HIF-1α/HIF-1ß protein-protein interaction to show the presence of an epigenetically controlled transactivation loop whereby the HIF-1 transcription factor promotes the expression of its own α-subunit in hypoxic cancer cells.

Biodegradable poly(α-hydroxy ester) blended microspheres as suitable carriers for retinal pigment epithelium cell transplantation.

Five blends of poly(L-lactic acid) (PLLA) with poly(D,L-lactic-co-glycolic acid) (PLGA) were used to prepare microcarriers by a solvent evaporation technique. Microcarriers were evaluated as suitable scaffolds to facilitate retinal pigment epithelium (RPE) cell transplantation. The blend ratios were 90:10, 75:25, 50:50, 25:75, 10:90 (PLLA:PLGA). Samples of each microcarrier blend were coated with an extracellular matrix protein. Coated and uncoated microcarriers were seeded with a human RPE cell line. As the lactide unit content increased the size of microcarriers generally became larger and the surface more irregular. Cells remained proliferative and retained phenotype as confirmed by immunocytochemistry. Blends rich in PLLA were superior for maintenance of RPE cell viability. This study demonstrates for the first time the feasibility of using microcarriers as a vehicle for retinal cell transplantation for ocular disease.

Rotor in a cage: Infrared spectroscopy of an endohedral hydrogen-fullerene complex.

We report the observation of quantized translational and rotational motion of molecular hydrogen inside the cages of C(60). Narrow infrared absorption lines at the temperature of 6 K correspond to vibrational excitations in combination with translational and rotational excitations and show well-resolved splittings due to the coupling between translational and rotational modes of the endohedral H(2) molecule. A theoretical model shows that H(2) inside C(60) is a three-dimensional quantum rotor moving in a nearly spherical potential. The theory provides both the frequencies and the intensities of the observed infrared transitions. Good agreement with the experimental results is obtained by fitting a small number of empirical parameters to describe the confining potential, as well as the relative concentration of ortho- and para-H(2).

Quantum translator-rotator: inelastic neutron scattering of dihydrogen molecules trapped inside anisotropic fullerene cages.

We report an inelastic neutron scattering investigation of the quantum dynamics of hydrogen molecules trapped inside anisotropic fullerene cages. Transitions among the manifold of quantized rotational and translational states are directly observed. The spectra recorded as a function of energy and momentum transfer are interpreted in terms of the rotational potential and the cage dimensions. The thermodynamics of orthohydrogen and parahydrogen are investigated through temperature dependence measurements.

Thermal history effects and methyl tunneling dynamics in a supramolecular complex of calixarene and para-xylene.

The low-temperature structure and dynamics of guest molecules of p-xylene incorporated in the isopropyl-calix[4] arene(2:1) p-xylene complex have been investigated by solid state nuclear magnetic resonance (NMR). Using one-dimensional 1H-decoupled 13C cross-polarization magic-angle-spinning (MAS) NMR and two-dimensional 1H-13C correlation spectroscopy, a full assignment of the 13C and 1H chemical shifts has been made. Using 1H NMR relaxometry, the effects of thermal history on the structure of the system have been investigated. Rapidly cooled samples have 1H spin-lattice relaxation times T1, which at low temperature (T<60 K) are typically two orders of magnitude faster than those observed in annealed samples which have been cooled slowly over many hours. In both forms, the low-temperature relaxation is driven by the dynamics of the weakly hindered methyl rotors of the p-xylene guest. The substantial difference in T1 is attributed in the rapidly cooled sample to disorder in the structure of the complex leading to a wide distribution of correlation times and methyl barrier heights. A comparison of the linewidths and splittings in the high resolution 13C MAS spectra of the two forms provides structural insight into the nature of the disorder. Using 1H field-cycling NMR relaxometry, the methyl dynamics of the p-xylene guest in the annealed sample have been fully characterized. The B-field dependence of the 1H T1 maps out the spectral density from which the correlation times are directly measured. The methyl barrier heights are determined from an analysis of the temperature dependence.

Solid-state NMR of endohedral hydrogen-fullerene complexes.

We present an overview of solid-state NMR studies of endohedral H(2)-fullerene complexes, including (1)H and (13)C NMR spectra, (1)H and (13)C spin relaxation studies, and the results of (1)H dipole-dipole recoupling experiments. The available data involves three different endohedral H(2)-fullerene complexes, studied over a wide range of temperatures and applied magnetic fields. The symmetry of the cage influences strongly the motionally-averaged nuclear spin interactions of the endohedral H(2) species, as well as its spin relaxation behaviour. In addition, the non-bonding interactions between fullerene cages are influenced by the presence of endohedral hydrogen molecules. The review also presents several pieces of experimental data which are not yet understood, one example being the structured (1)H NMR lineshapes of endohedral H(2) molecules trapped in highly symmetric cages at cryogenic temperatures. This review demonstrates the richness of NMR phenomena displayed by H(2)-fullerene complexes, especially in the cryogenic regime.

Cellular and molecular effects of protons: apoptosis induction and potential implications for cancer therapy.

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.