Ultrasound-Activated Microbubble Cancer Therapy: Ceramide Production Leading to Enhanced Radiation Effect in vitro

Abstract

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.

Ultrasound-stimulated microbubble enhancement of radiation response

“…This proof of principle research has been demonstrated in vitro Nofiele et al, 2013) and in vivo in a prostate cancer model (Czarnota et al, 2012a,b), in an in vivo bladder cancer model , and using a breast cancer model (unpublished results).…”

“…Exposure parameters are similar to that used with diagnostic ultrasound scans, with the exception that greater quantities of microbubbles are used to elicit an effect. On their own, the ultrasound effects described here result in minimal damage from which tissues recover but, when combined with radiation, result in striking amounts of cell death in tumours (AlMahrouki et al, 2012;Czarnota et al, 2012a,b;Tran et al, 2012;Nofiele et al, 2013).…”

“…All cell types in this study demonstrated ceramide immunohistochemical labelling in response to ultrasound-stimulated microbubble treatment, radiation, and more with the combination of the two treatment modalities. Asmase -/-cells showed little or no ceramide production with the exception of cells treated with ultrasound-stimulated microbubbles and radiation (Nofiele et al, 2013).…”

Photoacoustic spectral analysis to sense programmed erythrocyte cell death (eryptosis) for monitoring cancer response to treatment

“…The experiments were conducted using packed RBCs donated by healthy volunteers recruited by the Canadian Blood Services' Network Center for Applied Development (Vancouver, BC, CA). 1 ml of control blood sample and 1 ml of blood with 50 µM of C2-ceramide (Sigma Aldrich, ON, CA) were prepared to induce eryptosis [5]. The two vials were kept in an incubator at 37 o C and 280 RPM using Incu-Shaker (Benchmark Scientific, NJ, USA).…”

“…Microbubble shell disruption and gas release induces damage to the endothelial cells lining the vessels, thus resulting in subsequent vessel collapse [3], [4]. The damaged endothelial cells undergo apoptosis which have been shown to rapidly produce ceramide in the process [5]. Ceramide and phosphocholine are produced through the hydrolysis of sphingomyelin (part of the phospholipid bilayer) by acid sphingomyelinase (ASMases).…”