Sonodynamic Therapy Inhibits Angiogenesis And Tumor Growth

Objective: Angiogenesis is critical to tumor growth, metastasis, and recurrence. Sonodynamic therapy(SDT), based on the synergistic effects of ultrasound and sonosensitizers, is a promising approach for cancer treatment. Studies of SDT have mainly focused on its direct cytotoxic effect on tumor cells. Its effects on the tumor microenvironment, especially angiogenesis, remain unknown. The purpose of the present study is to examine the anti-angiogenic potency of SDT in vitro and in vivo.Methods: As a precursor of the sonosensitizer protoporphyrin IX, 5-aminolevulinic acid(ALA) was used in this study. We first analyzed inhibitory effect of SDT on human umbilical vein endothelial cells(HUVECs) in vitro. Cell proliferation was examined by MTT assay and trypan blue exclusion test. Cell migration and invasion were assessed by Transwell assay. Capillary-like tube formation was studied with a Matrigel model. A human tongue cancer SAS xenograft mouse model was used to examine the in vivo anti-angiogenic activity of SDT. Immunohistochemistry was used to evaluate microvessel density(MVD) and the expression of vascular endothelial growth factor(VEGF) in tumors. ELISA was used to measure the the secretion of VEGF in SAS cells. Ultrastructural changes in tumors were observed by transmission electron microscopy. Western blotting and semi-quantitative RT-PCR were used to determine the expression of intercellular adhesion molecule-1(ICAM-1).Results: By MTT assay and trypan blue exclusion test, inhibitory effect of the combination of ultrasound and ALA was observed on HUVEC proliferation(P<0.05). Examined with Transwell assay, HUVEC migration was more efficiently suppressed by combination of ultrasound and ALA than by ultrasound alone(P<0.05). The combination treatment of ultrasound and ALA also inhibited the invasion of HUVECs(P<0.05). Tube formation assay indicated that the anti-angiogenic effect of ultrasound was significantly enhanced by ALA(P<0.05). In a tumor xenograft mouse model, ultrasound plus ALA presented more potent efficacy in tumor growth suppression than the treatment of ultrasound alone(P<0.05). The combination treatment significantly inhibited the intratumoral vascularity compared with ultrasound alone(P<0.05). As a critical pro-angiogenic factor, the expression level of VEGF was reduced in tumors treated with SDT(P<0.05), which tended to coincident with the secretion of VEGF in SAS cells as determined by ELISA(P<0.05). An ultrastructural study showed damage and disruption of tumor microvasculature after SDT. In addition to inhibiting angiogenesis, SDT also enhances the expression of ICAM-1 on endothelial cells at both protein and mRNA levels(P<0.05).Conclusions: The above confirms the synergism of ultrasound and ALA on anti-angiogenesis and suppression of tumor growth. SDT might be a potent strategy for anticancer therapy. Anti-angiogenic potency of SDT is partially responsible for the anti-tumor effect of SDT.