Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism

<div><p>Ultrasound (US) stimulated microbubbles (MB) is a new treatment approach that sensitizes cancer cells to radiation (XRT). The molecular pathways in this response remain unelucidated, however, previous data has supported a role for cell membrane-metabolism related pathways including an up regulation of UDP glycosyltransferase 8 (UGT8), which catalyzes the transfer of galactose to ceramide, a lipid that is associated with the induction of apoptotic signalling. In this study, the role of UGT8 in responses of prostate tumours to ultrasound-stimulated microbubble radiation enhancement therapy is investigated. Experiments were carried out with cells <i>in vitro</i> and tumours <i>in</i> vivo in which UGT8 levels had been up regulated or down regulated. Genetically modified PC3 cells were treated with XRT, US+MB, or a combination of XRT+US+MB. An increase in the immunolabelling of ceramide was observed in cells where UGT8 was down-regulated as opposed to cells where UGT8 was either not regulated or was up-regulated. Clonogenic assays have revealed a decreased level of cellular survival with the down-regulation of UGT8. Xenograft tumours generated from stably transfected PC3 cells were also treated with US+MB, XRT or US+MB+XRT. Histology demonstrated more cellular damage in tumours with down-regulated UGT8 in comparison with control tumours. In contrast, tumours with up-regulated UGT8 had less damage than control tumours. Power Doppler imaging indicated a reduction in the vascular index with UGT8 down-regulation and photoacoustic imaging revealed a reduction in oxygen saturation. This was contrary to when UGT8 was up regulated. The down regulation of UGT8 led to the accumulation of ceramide resulting in more cell death signalling and therefore, a greater enhancement of radiation effect when vascular disruption takes place through the use of ultrasound-stimulated microbubbles.</p></div

Photoacoustic post-treatment images of xenograft PC3 modified tumours in vivo and oxygen saturation analyses.

<p>Percent change in oxygen saturation levels twenty-four hours after treatments is significantly different with the combined treatments, but not with the single treatments of sham, down regulated and up regulated tumours, where a reduction in the saturation is observed, with more reduction in the down regulated xenograft. Scale bar represents 1 mm.</p

Summary of UGT8 signalling and its effect on ceramide metabolism.

<p>When UGT8 levels are reduced, ceramide, either produced at the cellular membrane or in the endoplasmic reticulum, accumulates or initiates an apoptotic signal. However, when UGT8 levels are elevated, it adds a sugar moiety to ceramide in the Golgi apparatus, converting it to galactosylceramide, and this leads to the reduction of ceramide levels and possibly its apoptotic signalling, thus making cells more resistant to therapy.</p

H & E stained sections from xenograft PC3 modified tumours not treated with combinations of MB/XRT.

<p>The upper two panels show low magnification whole tumour sections and the lower two panels illustrate high magnification images. Controls, not transfected, or transfected with scrambled shRNA (sham) demonstrated apoptotic cell death more with combined treatments and less with single treatments. However, tumours induced by cells stably transfected with anti-<i>UGT8</i> sh-RNA, show different types of cell death and fibrosis. Cell death was less obvious in xenograft tumours initiated from cells with up-regulated <i>UGT8</i>. Scale bar for low magnification images is 1mm and for high magnification represents 50 μm.</p

TUNEL staining of xenograft PC3 modified tumours sections treated with various conditions.

<p>Control and sham tumours illustrate an increase in levels of apoptotic cell death which was significant in the combined treatments. A further increase in cell death is observed in tumours induced by cells stably transfected with anti-<i>UGT8</i> sh-RNA. In contrast, less cell death is observed with up-regulated <i>UGT8</i>. Scale bar represents 50 μm.</p

Transfection of PC3 cells <i>in vitro</i> and ceramide trafficking.

<p>Transfected cells were identified by the fluorescence of GFP. A) Transient transfection, showing fewer transfected cells than in the stably transfected cells. The levels of the <i>UGT8</i> gene and the UGT8 protein were evaluated after the stable transfection using RT-PCR, and ELISA, which verified the change in the levels of regulation. B) Ceramide trafficking in live images of cultured PC3 cells using BODIPY TR-labeled C₅ ceramide (red fluorescence), nuclear position is indicated by the nucleic acid stain Hoechst 33258 (blue fluorescence) with green fluorescence indicating transfection of constructs. After 3 h, control and down regulated cells demonstrtaed a higher level of ceramide uptake (red) that masks the green fluorescence compared to the cells over expressing UGT8. After 24 h, control and down regulated cells retained the exogenous ceramide (red) contrary to the cells over expressing <i>UGT8</i>. Scale bars represent 50 μm.</p

Ultrasound images of xenograft PC3 modified tumours in vivo and power Doppler analyses post-treatment.

<p>Vascular index change in vasculature 24 hours after treatments is significantly different with the combined treatments of either sham or down regulated tumours, where a reduction in the vascular index is observed, with more reduction in the down regulated xenograft. Although there is a reduction in the vasculature of the up-regulated tumours as well, it is not statistically significant. Scale bar represents 1 mm.</p

Ceramide, UGT8 labelling, and cellular survival after treatments.

<p>A) Control cells demonstrating increased levels of ceramide stained under various treatment conditions (brown, arrow). More condensed brown labelling was observed in down regulated cells under various treatments (arrow). In contrast, cells with up regulated UGT8, that were also subjected to similar conditions, demonstrate less ceramide labeling (arrows). B) UGT8 labelling was most intense in the up-regulated cells compared to the control or to the down-regulated cells (arrows). Scale bar represents 25 μm. C) Clonogenic assays indicate that control and up-regulated cells were less susceptible to treatment when compared to cells stably transfected with <i>UGT8</i> inhibitory shRNA.</p

Photoacoustic post-treatment images of xenograft PC3 modified tumours in vivo and oxygen saturation analyses.

<p>Percent change in oxygen saturation levels twenty-four hours after treatments is significantly different with the combined treatments, but not with the single treatments of sham, down regulated and up regulated tumours, where a reduction in the saturation is observed, with more reduction in the down regulated xenograft. Scale bar represents 1 mm.</p