Introduction

Ultrasound is widely used for soft tissue imaging because of its perceived safety, noninvasiveness and low cost. It is also used in therapy, which has shown effect on the suppression of the proliferation of bacteria, the improvement of the therapeutical effect of the drug, and in thrombolysis in vitro and so on (1-3). This effect of ultrasound can be strengthened by microbubbles (4,5). Microbubble is a blood contrast medium, and it can not be permeated to the outside of the blood vessel. For that reason, microbubble can be used in the ultrasound examination to observe the blood stream information of the organs, and large or small vessels. The diameter of the common microbubble is from 2 to 6 µm, which is similar to that of the red cell. After jet injection from the peripheral vein, and getting into the body, microbubble can pass pulmonary circulation and go into circulation system, also can strengthen the imaging of the organ (6,7). Cell permeabilization using microbubbles (MB) and ultrasound (US) have the potential of delivering molecules into the cytoplasm. The collapsing MB and cavitation bubbles created by this collapse generate impulsive pressures that cause transient membrane permeability, allowing exogenous molecules to enter the cells. Collapsed MB or cavitation bubbles generated by collapsed MB induce impulsive pressures such as liquid jets and shock waves, and these pressures affect the neighboring cells. The shock wave propagation distance from the center of a cavitation bubble that has the potential to damage the cell membrane is considerably larger than the maximum radius of the cavitation bubble (8). Several generations of the microbubble agents have also been developed. Early microbubbles contained an air core and were stabilized by a coating of albumin, starting with AlbunexR. Agents with a fluorinated gas core were then developed, including Optison^TM with a protein shell and perfluoropropane gas core and Definity^R with a phospholipid shell and perfluoropropane core. Microbubbles are typically manufactured by mechanical agitation, although microfluidic methods to engineer precise size distributions are in development (9). Cancer cells are more susceptible than normal cells to Sonodynamic therapy (SDT) (10,11), which serves as the experimental foundation for the application of SDT to the treatment of cancer. Recently SDT has been widely used in the therapy of cancer and has shown the effect of mediating apoptosis in many experimental systems in vitro or in vivo, but the detailed mechanism of this process is unclear. Moreover, the effect of ultrasound-induced apoptosis could be enhanced by porphyrin, anticancer drugs and other chemical compounds. The synergistic effect of SDT and other chemical compounds are referred to as sonodynamic therapy. In this review we will discuss the mechanism of the induction of the apoptosis of cancer cell by SDT.

Mechanism

Blood vessels of cancer were influenced by SDT

Angiogenesis, the process by which the existing vascular network expands to form new blood vessels, is required for the growth of solid tumors (12). Angiogenesis, the development of new blood vessels from the endothelium of a pre-existing vasculature, is a critical process required by most solid tumors to support their growth and metastasis. Therefore, anti-angiogenic therapy has been demonstrated to be an attractive strategy for cancer treatment. SDT could influence the vascular to induce cancer cell apoptosis in vivo (13). SDT combined with microbubbles also has effect on the vascular of cancer. Because the microbubbles are compressible, they alternately contract and expand in the acoustic field, a phenomenon referred to as cavitation. At low peak negative acoustic pressures are usually less than 0.2 MPa. As a result, microbubbles usually grow and shrink rhythmically and symmetrically around their equilibrium size, which is a phenomenon known as stable cavitation. At higher acoustic pressures, typically greater than 0.60 MPa, however, the expansion and contraction of microbubbles usually become unequal and markedly exaggerated, leading to vessel destruction. This activity is termed inertial cavitation, which induced the improvement of the cells membrane permeability and angiorrhexis of small vessels (14). When microbubbles are irradiated by ultrasound, they may induce the destruction of the vascular, and that of the vascular endothelium, causing thrombopoiesis in the vessels. It blocked the blood supply of the malignant tumor to induce the cancer apoptosis (15). Another study has found that SDT can facilitate anti-angiogenic gene delivery and inhibit prostate tumor growth in vitro and in vivo (16,17). Since glucose, oxygen, and other requirements are not evenly delivered through the tumor vasculature, the blood vessels develop and harbor hypoxic regions, the cells undergo oxidative stress and the vessels fail to mature, inducing the apoptosis of cancer cell (18).

SDT induced the apoptosis of cancer cell through the influence of the genes that have correlation with apoptosis

Modulating the expression of key molecular components of the apoptotic processes that comprise cell death is an attractive antineoplastic approach. In some experiments, it was found that SDT could influence the gene expression to induce apoptosis. In a study, human myelomonocytic lymphoma cell line U937 cells were exposed to the frequency of 1.0 MHz with 100 Hz pulse repetition frequency ultrasound. After that, cell viability and apoptosis, and gene expression were analyzed. This study showed that SDT could induce apoptosis, and down-regulate 193 genes and up-regulate 201 genes. For down-regulated genes, the significant genetic network was associated with cellular growth and proliferation, gene expression, or cellular development. For up-regulated genes, the significant genetic network was associated with cellular movement, cell morphology, and cell death. The present results indicate that SDT affect the expression of many genes and will provide novel insight into the bio-molecular mechanisms of SDT in therapeutic application for cancer therapy (19).

SDT could also improve gene transfection to therapy cancer and induce cancer cell apoptosis. Survivin, a member of the mammalian inhibitor of apoptosis protein (IAP) family, possesses multiple functions, including apoptosis inhibition, proliferation, tumorigenesis, cell cycle promotion (20). In all in vitro and in vivo experiments, it was found that ultrasound with microbubbles could improve survivin gene transfection, and could induce more of the apoptosis than that of the control group (21,22). Silencing of survivin gene expression with shRNA could be facilitated by this non-viral technique, and lead to significant cell apoptosis. This novel method for RNA interference represents a powerful and promising non-viral technology that can be used in the tumor gene therapy and research.

SDT influences the genes which are apoptotic. There are two main apoptotic pathways: the extrinsic (receptor-mediated) and the intrinsic (mitochondria-mediated). The intrinsic pathway of apoptosis may be triggered by both internal and external stimuli, including many mediators, which either promote or inhibit the process (23). The most representative regulators of the mitochondria- mediated pathway are p53, an inducer of apoptosis, and bcl-2, a molecule with the opposite function (24-26). In a study, it was found that protein 53, Bcl-2 were involved in ultrasound-induced apoptosis. Apoptosis and G1 arrest were induced primarily in p53⁺ cells, while p53^- cells showed less apoptosis but exhibited G₂ arrest. Likewise, the proliferation of cancer cells were much more strongly inhibited in p53⁺ than in p53^- cells (27), and Bcl-2 were shown to respond to ultrasound irradiation (28).

SDT could influence the cell signal pathway of cancer cell

Cell signal pathway plays an important role in the apoptosis of the cancer cell. The mitochondria-caspase signaling pathway was activated in the SDT-induced apoptosis of cancer cell, and ultrasound promotes the expression of pro-apoptotic proteins such as Bax and caspase-3 in cancer cell (29). In another study, the significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggested that active species such as singlet oxygen are important in the sonodynamic induction of apoptosis (30). Apoptosis induction has been reported to occur through a partial mediation of a Ca²⁺ dependent pathway. Calcium also has effect in the SDT induced cancer cell apoptosis. SDT can influence the cell ion pathway to induce apoptosis. Intracellular Ca²⁺ levels can vary depending on the bubble activity (31), which may be related to cell membrane damage and the amount of time required to repair this damage. Furthermore, some sonicated cells retain high levels of Ca²⁺ long after ultrasound exposure, which indicates a complete loss of cell membrane eintegrity (32). Ca²⁺ can improve the cell apoptosis level (33).

Drug chemotherapy could enhance the SDT induced cancer cell apoptosis

The drug chemotherapy is a very important method in the therapy of cancer (34). We constantly study how to enhance the sensitivity of the drug to reduce the therapeutic dose of the drug, and reduce the toxic and side-effect of the chemo-therapeutic drug. Ultrasound increases the membrane permeability without causing complete cell destruction which provides the experimental foundation for the enhancement of the drug osmosis of SDT in the treatment of cancer (35). In vitro study, MCF-7 cells were treated with 5-FU and sonicated at the frequency of 3.0 MHz and intensity of 3.0 W/cm² for 1 min in the presence of Optison. Immediately after the treatment, cell death was mostly dependent on Optison, however, 24 h after treatment, cell death was more dependent on 5-FU. Ultrasound duty cycle increased cell death associated with either Optison or 5-FU. Furthermore, the study showed that the treatment with 5-FU and ultrasound increase the levels of the Bax and p27^kip1 proteins (36). These studies not only showed that ultrasound can enhance the chemo-therapeutic effect in vitro but also studied the mechanism of it.

The other studies in vitro and in vivo also showed the SDT enhance the drug chemo-therapeutic effect of induction cancer cell apoptosis (37,38). These findings showed that ultrasound exposure was a promising technique for cancer chemotherapy.

SDT reversed of chemo-drug resistance of the cancer cell

Experimental and clinical investigations demonstrate that the chemotherapy induced toxic effect and the development of drug-resistance are the main barriers to successful therapies (39,40). Investigators hope to overcome drug-resistance, whilst maintaining or even improving the therapeutic effects. Ultrasound exposure could make drug resistance cancer cells more sensitive to anticancer drugs, which is a noninvasive physical approach for the induction of chemo-drug resistance reversal in cancer cells. In a study, they showed that the ultrasound effect on multidrug resistance (MDR) cancer cell. They investigated the mechanisms of therapeutic ultrasound as a physical approach to overcoming MDR in a multidrug resistant human hepato-carcinoma cell line (HepG₂/ADM). Their results demonstrated that ultrasound could reverse the chemo-drug resistance. Using RT-PCR technique, they found that US could significantly down-regulate the expression of P-glycoprotein (P-gp) and (MRP) at the mRNA level in HepG₂/ADM cells (41).Through this study, it not only confirmed that the SDT could reverse the chemo-drug resistance of the cancer cell but also found its mechanism. The study provided the experimental foundation for the clinical application of SDT combination with drug to the treatment of drug-resistance cancer.

Microbubble could enhance the SDT effect on inducing cancer cell apoptosis together with drugs

One recent approach targeting solid tumors is the application of microbubbles, which loaded with chemotherapeutic drugs. These advanced drug carriers could be safely administered to the patient by intravenous infusion, and would circulate through the entire vasculature. Their drug load could be locally released by ultrasound targeted microbubble destruction. In addition, tumors could be precisely localized by diagnostic ultrasound since microbubbles act as contrast agents (42). Microbubble combined with ultrasound could release the drug in specific position to save the drug and reduce the toxic and side-effect of the drug (43).

Summary

SDT can induce the apoptosis of cancer cell, and SDT permeabilizes the cell membrane directly, thereby allowing the delivery of exogenous molecules into the cells (44). It could also make bio-effects through physical methods, which gives us a new method to cure cancer.

Future direction

SDT has been widely used in the therapy of the cancer, and has got curative effect. Microbubbles combined with ultrasound have showed superiority in the therapy of the cancer. Especially microbubbles together with drugs or genes can even cure the cancer. However, the mechanisms of that are not yet clear, and in the future, it still needs to be studied. The best concentration of microbubbles and the frequency of the ultrasound in the treatment also need to be studied. Therefore, a better microbubble that can cure cancer together with drugs or genes is required. Furthermore, it must be stable and exhibit high performance in the delivery of the drugs or genes. The mechanisms of the apoptosis which SDT induces in the cancer cell in vitro, whether it still occurs in vivo or not, also need to be studied. We think that in the future SDT will be used more effectively in the treatment of cancer.

Acknowledgements

This study is supported by National Natural Science Foundation of China under grant No.81271597 and major infrastructure projects of Shanghai Science and Technology under grant No. 10JC1412600.

Disclosure: The authors declare no conflict of interest.

References

1. Conner-Kerr T, Alston G, Stovall A, et al. The effects of low-frequency ultrasound (35 kHz) on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. Ostomy Wound Manage 2010;56:32-43.
2. Maruani A, Boucaud A, Perrodeau E, et al. Low-frequency ultrasound sonophoresis to increase the efficiency of topical steroids: a pilot randomized study of humans. Int J Pharm 2010;395:84-90.
3. Tsivgoulis G, Eggers J, Ribo M, et al. Safety and efficacy of ultrasound-enhanced thrombolysis: a comprehensive review and meta-analysis of randomized and nonrandomized studies. Stroke 2010;41:280-7.
4. Sever A, Broillet A, Schneider M, et al. Dynamic visualization of lymphatic channels and sentinel lymph nodes using intradermal microbubbles and contrast-enhanced ultrasound in a swine model and patients with breast cancer. J Ultrasound Med 2010;29:1699-704.
5. Zhong H, Li R, Hao YX, et al. Inhibition effects of high mechanical index ultrasound contrast on hepatic metastasis of cancer in a rat model. Acad Radiol 2010;17:1345-9.
6. Mattrey RF, Aguirre DA. Advances in contrast media research. Acad Radiol 2003;10:1450-60.
7. Morawski AM, Lanza GA, Wickline SA. Targeted contrasts agents for magnetic resonance imagingand and ultrasound. Curr Opin Biotechnol 2005;16:89-92.
8. Kodama T, Tomita Y, Koshiyama K, et al. Transfection effect of microbubbles on cells in superposed ultrasound waves and behavior of cavitation bubble. Ultrasound Med Biol 2006;32:905-14.
9. Qin S, Caskey CF, Ferrara KW. Ultrasound contrast microbubbles in imaging and therapy: physical principles and engineering. Phys Med Biol 2009;54:R27-57.
10. Ashush H, Rozenszajn LA, Blass M, et al. Apoptosis induction of human myeloid leukemic cells by ultrasound exposure. Cancer Res 2000;60:1014-20.
11. Rosenthal I, Sostaric JZ, Riesz P. Sonodynamic therapy--a review of the synergistic effects of drugs and ultrasound. Ultrason Sonochem 2004;11:349-63.
12. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27-31.
13. Wood AK, Bunte RM, Price HE, et al. The disruption of murine tumor neovasculature by low-intensity ultrasound-comparison between 1- and 3-MHz sonication frequencies. Acad Radiol 2008;15:1133-41.
14. Shi WT, Forsberg F, Vaidyanathan P, et al. The influence of acoustic transmit parameters on the destruction of contrast microbubbles in vitro. Phys Med Biol 2006;51:4031-45.
15. Hwang JH, Brayman AA, Reidy MA, et al. Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treatments in vivo. Ultrasound Med Biol 2005;31:553-64.
16. Duvshani-Eshet M, Benny O, Morgenstern A, et al. Therapeutic ultrasound facilitates antiangiogenic gene delivery and inhibits prostate tumor growth. Mol Cancer Ther 2007;6:2371-82.
17. Nie F, Xu HX, Lu MD, et al. Anti-angiogenic gene therapy for hepatocellular carcinoma mediated by microbubble-enhanced ultrasound exposure: an in vivo experimental study. J Drug Target 2008;16:389-95.
18. Gordon MS, Mendelson DS, Kato G. Tumor angiogenesis and novel antiangiogenic strategies. Int J Cancer 2010;126:1777-87.
19. Tabuchi Y, Takasaki I, Zhao QL, et al. Genetic networks responsive to low-intensity pulsed ultrasound in human lymphoma U937 cells. Cancer Lett 2008;270:286-94.
20. Ai Z, Yin L, Zhou X, et al. Inhibition of survivin reduces cell proliferation and induces apoptosis in human endometrial cancer. Cancer 2006;107:746-56.
21. Chen Z, Liang K, Xie M, et al. Novel ultrasound-targeted microbubble destruction mediated short hairpin RNA plasmid transfection targeting survivin inhibits gene expression and induces apoptosis of HeLa cells. Mol Biol Rep 2009;36:2059-67.
22. Chen ZY, Liang K, Qiu RX. Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis. J Exp Clin Cancer Res 2010;29:152.
23. Park JW, Ryter SW, Choi AM. Functional significance of apoptosis in chronic obstructive pulmonary disease. COPD 2007;4:347-53.
24. Schuler M, Green DR. Mechanisms of p53-dependent apoptosis. Biochem Soc Trans 2001;29:684-8.
25. Haupt S, Berger M, Goldberg Z, et al. Apoptosis - the p53 network. J Cell Sci 2003;116:4077-85.
26. Martin DA, Elkon KB. Mechanisms of apoptosis. Rheum Dis Clin North Am 2004;30:441-54, vii.
27. Abdollahi A, Domhan S, Jenne JW, et al. Apoptosis signals in lymphoblasts induced by focused ultrasound. FASEB J 2004;18:1413-4.
28. Feng Y, Tian Z, Wan M. Bioeffects of low-intensity ultrasound in vitro: apoptosis, protein profile alteration, and potential molecular mechanism. J Ultrasound Med 2010;29:963-74.
29. Tang W, Liu Q, Zhang J, et al. In vitro activation of mitochondria-caspase signaling pathway in sonodynamic therapy-induced apoptosis in sarcoma 180 cells. Ultrasonics 2010;50:567-76.
30. Yumita N, Okudaira K, Momose Y, et al. Sonodynamically induced apoptosis and active oxygen generation by gallium-porphyrin complex, ATX-70. Cancer Chemother Pharmacol 2010;66:1071-8.
31. Juffermans LJ, Dijkmans PA, Musters RJ, et al. Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide. Am J Physiol Heart Circ Physiol 2006;291:H1595-601.
32. Kumon RE, Aehle M, Sabens D, et al. Spatiotemporal effects of sonoporation measured by real-time calcium imaging. Ultrasound Med Biol 2009;35:494-506.
33. Hutcheson JD, Schlicher RK, Hicks HK, et al. Saving cells from ultrasound-induced apoptosis: quantification of cell death and uptake following sonication and effects of targeted calcium chelation. Ultrasound Med Biol 2010;36:1008-21.
34. Kristensen GB, Tropé C. Epithelial ovarian carcinoma. Lancet 1997;349:113-7.
35. Korosoglou G, Hardt SE, Bekeredjian R, et al. Ultrasound exposure can increase the membrane permeability of human neutrophil granulocytes containing microbubbles without causing complete cell destruction. Ultrasound Med Biol 2006;32:297-303.
36. Chumakova OV, Liopo AV, Evers BM, et al. Effect of 5-fluorouracil, Optison and ultrasound on MCF-7 cell viability. Ultrasound Med Biol 2006;32:751-8.
37. Tian Z, Quan X, Xu C, et al. Hematoporphyrin monomethyl ether enhances the killing action of ultrasound on osteosarcoma in vivo. J Ultrasound Med 2009;28:1695-702.
38. Watanabe Y, Aoi A, Horie S, et al. Low-intensity ultrasound and microbubbles enhance the antitumor effect of cisplatin. Cancer Sci 2008;99:2525-31.
39. Armstrong DK. Relapsed ovarian cancer: challenges and management strategies for a chronic disease. Oncologist 2002;7:20-8.
40. Dunton CJ. Management of treatment-related toxicity in advanced ovarian cancer. Oncologist 2002;7:11-9.
41. Wu F, Shao ZY, Zhai BJ, et al. Ultrasound reverses multidrug resistance in human cancer cells by altering gene expression of ABC transporter proteins and Bax protein. Ultrasound Med Biol 2011;37:151-9.
42. Tinkov S, Winter G, Coester C, et al. New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: Part I--Formulation development and in-vitro characterization. J Control Release 2010;143:143-50.
43. Kang J, Wu X, Wang Z, et al. Antitumor effect of docetaxel-loaded lipid microbubbles combined with ultrasound-targeted microbubble activation on VX2 rabbit liver tumors. J Ultrasound Med 2010;29:61-70.
44. Bai WK, Wu ZH, Shen E, et al. The improvement of liposome-mediated transfection of pEGFP DNA into human prostate cancer cells by combining low-frequency and low-energy ultrasound with microbubbles. Oncol Rep 2012;27:475-80.