Search

Blood vessels within tumours represent a key component for cancer cell survival. Disruption of these vessels can be achieved by inducing vascular endothelial-cell apoptosis. Moreover, endothelial cell apoptosis has been proven to be enhanced by ceramide-increasing drugs. Herein, we introduce a novel therapeutic approach which uses ultrasound-stimulated microbubbles used in combination with radiation to cause a rapid accumulation of ceramide in endothelial cells in-vitro. We also test this modality directly with other cell types as a general method of killing cancer cells. Human umbilical vein endothelial cells (HUVEC), acute myeloid leukemia cells (AML), murine fibrosarcoma cells (KHT-C), prostate cancer cells (PC3), breast cancer cells (MDA-MB-231) and astrocytes were used to evaluate this mechanism of inducing cell death. Survival was measured by clonogenic assays, and ceramide content was detected using immunohistochemistry. Exposure of cell types to ultrasound-stimulated bubbles alone resulted in increases in ceramide for all cell types and survivals of 12 ± 2%, 65 ± 5%, 83 ± 2%, 58 ± 4%, 58 ± 3%, 18 ± 7% for HUVEC, AML, PC3, MDA, KHT-C and astrocyte cells, respectively. Results from selected cell types involving radiation treatments indicated additive treatment enhancements and increases in intracellular ceramide content one hour after exposure to ultrasound-activated microbubbles and radiation. Endothelial cell survival decreased from 8 ± 1% after 2 Gy of radiation treatment alone and from 12 ± 2% after ultrasound and microbubbles alone, to 1 ± 1% with combined treatment. In Asmase +/+ astrocytes, survival decreased from 56 ± 2% after 2 Gy radiation alone and from 17 ± 7% after ultrasound and microbubbles alone, to 5 ± 2% when combined. Using ASMase deficient astrocytes ( Asmase -/-) and Sphingosine-1-phosphate (S1P), we also demonstrate that ultrasound-activated microbubbles stimulate ASMase activity and ceramide production. These findings suggest that ultrasound-stimulated microbubbles could be used as a new biomechanical method to enhance the effects of radiation.

We have discovered that ultrasound-mediated microbubble vascular disruption can enhance tumor responses to radiation in vivo. We demonstrate this effect using a human PC3 prostate cancer xenograft model. Results indicate a synergistic effect in vivo with combined single treatments of ultrasound-stimulated microbubble vascular perturbation and radiation inducing an over 10-fold greater cell kill with combined treatments. We further demonstrate with experiments in vivo that induction of ceramide-related endothelial cell apoptosis, leading to vascular disruption, is a causative mechanism. In vivo experiments with ultrasound and bubbles permit radiation doses to be decreased significantly for comparable effect. We envisage this unique combined ultrasound-based vascular perturbation and radiation treatment method being used to enhance the effects of radiation in a tumor, leading to greater tumor eradication.

To improve our understanding of proteins interactions with cellular membranes, we have developed a method based on confocal microscopy that enable us to correlate the number of proteins bound to a lipid vesicle with the vesicle properties (membrane integrity, membrane potential, vesicle size¼). This method consists in the simultaneous recording of confocal images in two different channels, and in the coincidental detection of single mobile particles in these channels. To demonstrate the usefulness of this method, we have used a model system consisting of small unilamellar liposomes (∼ 100 nm in diameter) rendered fluorescent by the addition of a small percentage of fluorophore and interacting with fluorescently labeled proteins.To optimize image acquisition parameters for the detection of single diffusing liposomes, we systematically varied the fluorophore to vesicle ratio, the fluorescence excitation intensity, and the confocal pixel size and dwell time. We analyzed the images using different pixel binning values and different detection threshold. We then determined the range of parameters for which we were able to detect particles with independent positions. We also verified that the particle concentration and average particle fluorescence emission extracted from the images using this single particle detection method were consistent with results obtained by fluorescence correlation spectroscopy (FCS), raster-scanning image correlation spectroscopy (RICS) and fluorescence intensity distribution analysis (FIDA). Using vesicles labeled with two different fluorophores, we measured the probability that a vesicle detected in one channel would also be detected in the second channel, and compared our results with those obtained by fluorescence cross-correlation spectroscopy (XCS).Our method should be useful for studies of protein or peptide insertion and pore formation into lipid membranes.

Bcl-2 family proteins play a crucial role in the regulation of the permeabilization of the outer mitochondrial membrane (OMM), which is one of the main steps leading to cell death through apoptosis.To a first approximation, apoptosis is controlled by the balance between pro-apoptotic and anti-apoptotic Bcl-2 family proteins. Deregulation of this balance induces a dysfunction of the apoptosis process leading to different pathologies. The molecular mechanism leading to OMM permeabilization is not yet understood in details. This is in part because of the multiplicity and complexity of the molecular interactions involving Bcl-2 family proteins, since each of them can often interact with itself (homo-oligomerization), with other members of the family (hetero-oligomerization) and with the lipids of the OMM.Whereas in most studies ensemble methods have been used to study these proteins, we have used a single mobile particle detection method to characterize the distribution of different Bcl-2 family proteins on a population of large unilamellar liposomes, in order to better understand the interplay between different family members and lipid membranes during the process of membrane permeabilization. We used large unilamellar liposomes (∼ 200 nm in diameter) with a lipid composition mimicking that of mitochondrial membranes and fluorescently labeled recombinant proteins, which were imaged by confocal microscopy. The specific brightness of each particle detected in the images can then calculated for each separate emission channel.As a first application, we studied the distribution of Bax and Bid on a population of liposomes. These proteins are both pro-apoptotic, promoting pore formation in liposomes, and they are known to interact with one another. We measured the probability that a liposome carried Bax, Bid, or both proteins, allowing us to quantify their colocalization on the same liposome.