Journal of Medical Ultrasonics

, Volume 38, Issue 2, pp 65–72 | Cite as

Differential cytotoxicity and sonosensitization by sanazole: effect of cell type and acoustic parameters

  • Mariame A. Hassan
  • Yukihiro Furusawa
  • Qing-Li Zhao
  • Ichiro Takasaki
  • Loreto B. FerilJr.
  • Katsuro Tachibana
  • Nobuki Kudo
  • Masami Minemura
  • Toshiro Sugiyama
  • Takashi Kondo
Original Article



Although sanazole has been used as a hypoxic radiosensitizer, we recently reported on its ability to sensitize U937 cells to hyperthermia and X-irradiation under aerobic conditions, enhancing apoptotic cell death following the combined treatment. The current study was undertaken to evaluate the effect of sanazole as a sonosensitizer under previously studied acoustic conditions of different pulse repetition frequencies, using two cell lines representative of solid tumours and haematopoietic cancers.


Cells were treated with different doses of sanazole. Flow-cytometric analysis and DNA fragmentation assay were carried out at different times, and morphological features were also inspected. For ultrasound treatment, cells were pre-incubated with a non-cytotoxic dose of sanazole for 30 min before exposure. Evaluation of cell killing and a parallel examination of intracellular oxidative stress levels in both cell lines were performed using flow cytometry.


Sanazole alone displayed selective cytotoxic effects towards solid tumour-derived cancer cells, resulting in complete cell death after 24 h of treatment, and enhanced the ultrasound-induced cell killing 6 h post-treatment. The enhancement seemed to be mediated by an additive increase in intracellular oxidative stress levels.


Sanazole seems to be an efficient cytotoxic agent for the treatment of solid tumours and a promising sonosensitizer under aerobic conditions.


Apoptosis Pulse repetition frequency Sanazole Sonosensitizer Ultrasound 



Part of this work was supported by the Research and Development Committee Program of the Japan Society of Ultrasonics in Medicine, and by a Grant-in-Aid for Scientific Research (C) (No. 20590765) (to MM) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.


  1. 1.
    Shibamoto Y, Sakano K, Kimura R, Nishidai T, Nishimoto S, Ono K, Kagiya T, Abe M. Radiosensitization in vitro and in vivo by 3-nitrotriazoles. Int J Radiat Oncol Biol Phys. 1986;12:1063.PubMedCrossRefGoogle Scholar
  2. 2.
    Dobrowsky W, Huigol NG, Jayatilake RS, Kizilbash NI, Okkan S, Kagiya TV, Tatsuzaki H. AK-2123 (Sanazol) as a radiation sensitizer in the treatment of stage III cancer cervix: initial results of an IAEA multicentre randomized trial. J Cancer Res Ther. 2005;1:75.PubMedCrossRefGoogle Scholar
  3. 3.
    Dobrowsky W, Huigol NG, Jayatilake RS, Kizilbash NI, Okkan S, Kagiya VT, Tatsuzaki H. AK-2123 (Sanazol) as a radiation sensitizer in the treatment of stage III cervical cancer: results of an IAEA multicentre randomised trial. Radiother Oncol. 2007;82:24.PubMedCrossRefGoogle Scholar
  4. 4.
    Ullal SD, Shenoy KK, Pai MR, Chowta MN, Adiga SM, Dinesh M, Kamath A, Kotian MS, Pai DK. Safety and radiosensitizing efficacy of sanazole (AK 2123) in oropharyngeal cancers: randomized controlled double blind clinical trial. Indian J Cancer. 2006;43:151.PubMedCrossRefGoogle Scholar
  5. 5.
    Konovalova NP, Volkova LM, Tatyanenko LV, Kotelnikova RA, Yakushchenko TN, Kagiya TV. Inhibitory effect of radiosensitizer AK-2123 on experimental hepatic metastases and Ca2+ active transport. Neoplasma. 1997;44:361.PubMedGoogle Scholar
  6. 6.
    Konovalova NP, Diatchkovskaya RF, Volkova LM, Kagiya TV. Radiosensitizer AK-2123 as modulating agent in the chemotherapy of experimental metastases. Neoplasma. 1995;42:119.PubMedGoogle Scholar
  7. 7.
    Mitsuhashi N, Sakurai H, Takahashi T, Akimoto T, Higuchi K, Matsumoto H, Ebara T, Nozaki M, Niibe H. Does AK-2123 (Senazole) have sensitizing effects on radiation, cisplatin and hyperthermia under aerobic conditions in vitro? Anticancer Res. 1998;18:3463.PubMedGoogle Scholar
  8. 8.
    Sugie C, Shibamoto Y, Ito M, Ogino H, Suzuki H, Uto Y, Nagasawa H, Hori H. Reevaluation of the radiosensitizing effects of sanazole and nimorazole in vitro and in vivo. J Radiat Res (Tokyo). 2005;46:453.CrossRefGoogle Scholar
  9. 9.
    Feril LB Jr, Kondo T. Biological effects of low intensity ultrasound: the mechanism involved, and its implications on therapy and on biosafety of ultrasound. J Radiat Res (Tokyo). 2004;45:479.CrossRefGoogle Scholar
  10. 10.
    Yoshida T, Kondo T, Ogawa R, Feril LB Jr, Zhao QL, Watanabe A, Tsukada K. Combination of doxorubicin and low-intensity ultrasound causes a synergistic enhancement in cell killing and an additive enhancement in apoptosis induction in human lymphoma U937 cells. Cancer Chemother Pharmacol. 2008;61:559.PubMedCrossRefGoogle Scholar
  11. 11.
    Kondo T, Yoshida T, Ogawa R, Hassan MA, Furusawa Y, Zhao QL, Watanabe A, Morii A, Feril LB Jr, Tachibana K, Kitagawa H, Tabuchi Y, Takasaki I, Shehata M, Kudo N, Tsukada K. Low-intensity ultrasound adjuvant therapy: enhancement of doxorubicin-induced cytotoxicity and the acoustic mechanisms involved. J Med Ultrasonics. 2009;36:61.CrossRefGoogle Scholar
  12. 12.
    Hassan MA, Campbell P, Kondo T. The role of Ca2+ in ultrasound-elicited bioeffects: progress, perspectives, and prospects. Drug Discov Today. 2010;15:892.PubMedCrossRefGoogle Scholar
  13. 13.
    Honda H, Kondo T, Zhao QL, Feril LB Jr, Kitagawa H. Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound. Ultrasound Med Biol. 2004;30:683.PubMedCrossRefGoogle Scholar
  14. 14.
    Buldakov MA, Hassan MA, Zhao QL, Feril LB Jr, Kudo N, Kondo T, Litvyakov NV, Bolshakov MA, Rostov VV, Cherdyntseva NV, Riesz P. Influence of changing pulse repetition frequency on chemical and biological effects induced by low-intensity ultrasound in vitro. Ultrason Sonochem. 2009;16:392.PubMedCrossRefGoogle Scholar
  15. 15.
    Hassan MA, Buldakov MA, Ogawa R, Zhao QL, Furusawa Y, Kudo N, Kondo T, Riesz P. Modulation control over ultrasound-mediated gene delivery: evaluating the importance of standing waves. J Control Release. 2010;141:70.PubMedCrossRefGoogle Scholar
  16. 16.
    Feril LB Jr, Kondo T. Major factors involved in the inhibition of ultrasound-induced free radical production and cell killing by pre-sonication incubation or by high cell density. Ultrason Sonochem. 2005;12:353.PubMedCrossRefGoogle Scholar
  17. 17.
    Furusawa Y, Zhao QL, Hassan MA, Tabuchi Y, Takasaki I, Wada S, Kondo T. Ultrasound-induced apoptosis in the presence of sonazoid and associated alterations in gene expression levels: a possible therapeutic application. Cancer Lett. 2010;288:107.PubMedCrossRefGoogle Scholar
  18. 18.
    Wada S, Cui ZG, Kondo T, Zhao QL, Ogawa R, Shoji M, Arai T, Makino K, Furuta I. A hydrogen peroxide-generating agent, 6-formylpterin, enhances heat-induced apoptosis. Int J Hyperthermia. 2005;21:231.PubMedCrossRefGoogle Scholar
  19. 19.
    Li M, Kondo T, Zhao QL, Li FJ, Tanabe K, Arai Y, Zhou ZC, Kasuya M. Apoptosis induced by cadmium in human lymphoma U937 cells through Ca2+-calpain and caspase-mitochondria-dependent pathways. J Biol Chem. 2000;275:39702.PubMedCrossRefGoogle Scholar
  20. 20.
    Yoshihisa Y, Honda A, Zhao QL, Makino T, Abe R, Matsui K, Shimizu H, Miyamoto Y, Kondo T, Shimizu T. Protective effects of platinum nanoparticles against UV-light-induced epidermal inflammation. Exp Dermatol. 2010;19:1000–6.Google Scholar
  21. 21.
    Li FJ, Kondo T, Zhao QL, Tanabe K, Ogawa R, Li M, Arai Y. Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells. Free Radic Res. 2001;35:281.PubMedCrossRefGoogle Scholar
  22. 22.
    Ahmed K, Zhao QL, Matsuya Y, Yu DY, Feril LB Jr, Nemoto H, Kondo T. Rapid and transient intracellular oxidative stress due to novel macrosphelides trigger apoptosis via Fas/caspase-8-dependent pathway in human lymphoma U937 cells. Chem Biol Interact. 2007;170:86.PubMedCrossRefGoogle Scholar
  23. 23.
    Yu DY, Zhao QL, Wei ZL, Shehata M, Kondo T. Enhancement of hyperthermia-induced apoptosis by sanazole in human lymphoma U937 cells. Int J Hyperthermia. 2009;25:364.PubMedCrossRefGoogle Scholar
  24. 24.
    Yu DY, Zhao QL, Wei ZL, Nomura T, Kashiwakura I, Kagiya TV, Kondo T. Enhancement of radiation-induced apoptosis of human lymphoma U937 cells by sanazole. Apoptosis. 2009;14:655.PubMedCrossRefGoogle Scholar
  25. 25.
    Ohnuma T, Arkin H, Holland JF. Effects of cell density on drug-induced cell kill kinetics in vitro (inoculum effect). Br J Cancer. 1986;54:415.PubMedCrossRefGoogle Scholar
  26. 26.
    Smrz D, Draberova L, Draber P. Non-apoptotic phosphatidylserine externalization induced by engagement of glycosylphosphatidylinositol-anchored proteins. J Biol Chem. 2007;282:10487.PubMedCrossRefGoogle Scholar
  27. 27.
    Yan B, Wang H, Peng Y, Hu Y, Wang H, Zhang X, Chen Q, Bedford JS, Dewhirst MW, Li CY. A unique role of the DNA fragmentation factor in maintaining genomic stability. Proc Natl Acad Sci USA. 2006;103:1504.PubMedCrossRefGoogle Scholar
  28. 28.
    Kim G, Lee YE, Xu H, Philbert MA, Kopelman R. Nanoencapsulation method for high selectivity sensing of hydrogen peroxide inside live cells. Anal Chem. 2010;82:2165.PubMedCrossRefGoogle Scholar
  29. 29.
    Miller DL, Dou C. Induction of apoptosis in sonoporation and ultrasonic gene transfer. Ultrasound Med Biol. 2009;35:144.PubMedCrossRefGoogle Scholar
  30. 30.
    Imamura M, Edgren M, Murata T, Imamura M, Nagata K, Isoda H, Akagi K, Revesz L, Tanaka Y. Radiosensitization with a 3-nitrotriazole (AK-2123). Int J Oncol. 1995;6:841.Google Scholar
  31. 31.
    Ingber DE. Cellular mechanotransduction: putting all the pieces together again. FASEB J. 2006;20:811.PubMedCrossRefGoogle Scholar
  32. 32.
    Rojkind M, Dominguez-Rosales JA, Nieto N, Greenwel P. Role of hydrogen peroxide and oxidative stress in healing responses. Cell Mol Life Sci. 2002;59:1872.PubMedCrossRefGoogle Scholar
  33. 33.
    Kapoor S, Mathew R, Huilgol NG, Kagiya TV, Nair CK. Redox reactions of sanazole (AK-2123) in aqueous solutions: a pulse radiolysis study. J Radiat Res (Tokyo). 2000;41:355.CrossRefGoogle Scholar
  34. 34.
    Sukhanov GT, Sukhanova AG, Ilyasova YV. Reactions of 3-nitro-1,2,4-triazoles with alkylating agents. 3. Alkylation of a neutral heterocycle by diethyl sulfate. Chem Heterocycl Comp. 2006;42:1197.Google Scholar
  35. 35.
    Alam A, Rapthap CC, Singha LI, Sharan RN, Singh V. Radiomodulatory effect of liposome encapsulated AK-2123 on tumor in mice exposed to hepatocarcinogen. Mol Cell Biochem. 2005;271:139.PubMedCrossRefGoogle Scholar

Copyright information

© The Japan Society of Ultrasonics in Medicine 2010

Authors and Affiliations

  • Mariame A. Hassan
    • 1
    • 2
  • Yukihiro Furusawa
    • 1
  • Qing-Li Zhao
    • 1
  • Ichiro Takasaki
    • 3
  • Loreto B. FerilJr.
    • 4
  • Katsuro Tachibana
    • 4
  • Nobuki Kudo
    • 5
  • Masami Minemura
    • 6
  • Toshiro Sugiyama
    • 6
  • Takashi Kondo
    • 1
  1. 1.Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  2. 2.Department of Pharmaceutics and Industrial Pharmacy, Faculty of PharmacyCairo UniversityCairoEgypt
  3. 3.Division of Molecular Genetic Research, Life Science Research CenterUniversity of ToyamaToyamaJapan
  4. 4.Department of AnatomySchool of Medicine, Fukuoka UniversityFukuokaJapan
  5. 5.Laboratory of Biomedical Instrumentation and Measurement, Graduate School of Information Science and TechnologyHokkaido UniversitySapporoJapan
  6. 6.Department of Gastroenterology and Hematology, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan

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