Ultrasound-Stimulated Microbubbles Enhance Radiosensitization of Nasopharyngeal Carcinoma

Abstract

Background/Aims: Recent studies indicate that therapies targeting the vasculature can significantly sensitize tumors to radiation. Ultrasound-stimulated microbubbles (USMBs) are regarded as a promising radiosensitizer. In this study, we investigated the effect of USMBs on the sensitivity of nasopharyngeal carcinoma (NPC) to radiation. Methods: Human NPC (CNE-2) cells and human umbilical vein endothelial cells (HUVECs) were exposed to radiation (0, 2, and 8 Gy) alone or in combination with USMBs. Cell viability and apoptosis were measured with the MTT assay and flow cytometry, respectively. The angiogenic activity of HUVECs was detected using matrigel tubule formation. The in vitro effects induced by these treatments were confirmed in vivo with xenograft models of CNE-2 cells in nude mice by examining vascular integrity using color Doppler flow imaging and cell survival using immunohistochemistry. Additionally, the in vivo and in vitro expressions of angiotensin II (ANG II) and its receptor (AT1R) were detected by immunohistochemistry and western blotting, respectively. With CNE-2 cells and HUVECs transfected with control, ANG II, or AT1R, perindopril (an inhibitor of angiotensin-converting enzyme) and candesartan (an inhibitor of AT1R) were used to verify the role of ANG II and AT1R in the radiosensitivity of tumor and endothelial cells by USMBs, by determining cell viability and apoptosis and angiogenic activity. Results: In the NPC xenografts, USMBs slightly reduced blood flow and CD34 expression, increased tumor cell death and ANG II and AT1R expression, and significantly enhanced the effects of radiation. With CNE-2 cells and HUVECs, the USMBs further enhanced the inhibition of tumor cell viability and endothelial tubule formation and further enhanced the increase in ANG II and AT1R due to radiation. Furthermore, perindopril and candesartan significantly enhanced the inhibitory effect of radiation and USMBs on tumor cell growth and angiogenesis in vitro. Conclusions: We have demonstrated for the first time that USMB exposure can significantly enhance the destructive effect on NPC of radiation, and this effect might be further increased by ANG II and AT1R inhibition. Our findings suggest that USMBs can be used as a promising sensitizer of radiotherapy to treat NPC, and the clinical effect might be increased by ANG II and AT1R inhibition.

Ultrasound-Stimulated Microbubbles Enhance Radiation-induced Cell Killing

“…Deng et al [16] treated NCE-2 nasopharyngeal carcinoma cells with 3% v/v per uropropane-based USMBs, administered 24 hours prior to exposure to 2Gy or 8Gy RT. Cell viability was measured using the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay 24, 48 and 72 hours post-treatment.…”

“…Cell seeding densities and MTS development times were optimised to achieve 80% con uence at 72 hours post-treatment for each cell line, as summarised in Table 1. This level of con uence was chosen in order to avoid any potential effects of contact-dependent cell signalling or contact inhibition, and 72 hours is consistent with the manufacturer's instructions for cell proliferation studies and other experiments using this assay [21][22][23] or the MTT assay [16]. Tullamarine, Australia).…”

“…Treatment order and time between treatments also impacted clonogenic survival, with greatest decreases in clonogenic viability seen when PC3 cells were exposed to USMB followed by immediate 3Gy treatment (17%), compared to that of KHT-C cells treated with USMB followed by a 3-hour gap before being exposed to 3Gy (30%). Deng et al [16] treated NCE-2 nasopharyngeal carcinoma cells with 3% v/v per uropropane-based USMBs, administered 24 hours prior to exposure to 2Gy or 8Gy RT. Cell viability was measured using the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay 24, 48 and 72 hours post-treatment.…”

“…Cell Proliferation Assays were initially optimised in the absence of treatments using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (catalogue number G3582, Promega, Australia). The MTS tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is similar to the MTT compound used in previous studies [16], and is bioreduced by viable cells into a coloured formazan product that can be analysed via colorimetry. Cells are incubated with the reagent for between 1-4 hours, and then absorbance readings taken at 490nm in a 96-well plate reader.…”

“…Both ask sizes were then lled with 30ml of cell-line speci c complete media. De nity® Microbubbles (Lantheus Medical Imaging, Inc, supplied by Global Medical Solutions, Keilor Park, Australia) were activated using the Vialmix® activation device (Lantheus Medical Imaging, Inc) following the manufacturer's instructions [24], and 480µl added to the relevant treatment asks to give a nal microbubble concentration of 1.6% v/v, which was close to that described in other in vitro studies [11][12][13][14][15][16].…”