, Volume 12, Issue 8, pp 1523–1532 | Cite as

Enhancement of hyperthermia-induced apoptosis by a new synthesized class of furan-fused tetracyclic compounds

  • Da-Yong Yu
  • Yuji Matsuya
  • Qing-Li Zhao
  • Kanwal Ahmed
  • Zheng-Li Wei
  • Hideo Nemoto
  • Takashi KondoEmail author
Original Paper


The combined effects of hyperthermia (44°C, 20 min) or X-rays (10 Gy) and a new class of furan-fused tetracyclic synthesized compounds (DFs), on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (DF1∼6), the combined treatment of 10 μM DF with TIPS (triisopropylsilyloxy) (Designated #3 DF3) and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia but no enhancement was observed if it was combined with X-rays. Enhancement of hyperthermia-induced apoptosis by DF3 in a dose-dependent manner was observed. When the cells were treated first with DF3 at a nontoxic concentration of 20 μM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by hyperthermia, was enhanced by DF3. Mitochondrial transmembrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. Externalization of Fas was observed following the combined treatment. Flow cytometry revealed rapid and sustained increase of intracellular superoxide due to DF3, and showed subsequent and transient increase in the formation of intracellular hydrogen peroxide (H2O2), which was further increased when hyperthermia was combined. These results indicate that the intracellular superoxide and H2O2 generated by DF3 enhance the hyperthermia-induced apoptosis via the Fas-mediated mitochondrial caspase-dependent pathway.


Apoptosis Hyperthermia Reactive oxygen species 


  1. 1.
    Lee RH, Slate DL, Moretti R, Alvi KA, Crews P (1992) Marine sponge polyketide inhibitors of protein tyrosine kinase. Biochem Biophys Res Commun 184:765–772PubMedCrossRefGoogle Scholar
  2. 2.
    Roll DM, Scheuer PJ, Matsumoto GK, Clardy J (1983) Halenaquinone, a pentacyclic polyketide from a marine sponge. J Am Chem Soc 105:6177–6178CrossRefGoogle Scholar
  3. 3.
    Schmitz FJ, Bloor SJ (1988) Xesto- and halenaquinone derivatives from a sponge, Adocia sp., from truk lagoon. J Org Chem 53:3922–3925CrossRefGoogle Scholar
  4. 4.
    Fujiwara H, Matsunaga K, Saito M et al (2001) Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells. Eur J Pharmacol 413:37–45PubMedCrossRefGoogle Scholar
  5. 5.
    Matsuya Y, Sasaki K, Nagaoka M et al (2004) Synthesis of a new class of furan-fused tetracyclic compounds using o-quinodimethane chemistry and investigation of their antiviral activity. J Org Chem 69:7989–7993PubMedCrossRefGoogle Scholar
  6. 6.
    Harima Y, Nagata K, Harima K, Ostapenko VV, Tanaka Y, Sawada S (2001) A randomized clinical trial of radiation therapy versus thermoradiotherapy in stage IIIB cervical carcinoma. Int J Hyperthermia 17:97–105PubMedCrossRefGoogle Scholar
  7. 7.
    Overgaard J, Gonzalez Gonzalez D, Hulshof MC et al (1995) Randomised trial of hyperthermia as adjuvant to radiotherapy for recurrent or metastatic malignant melanoma. European Society for Hyperthermic Oncology. Lancet 345:540–543PubMedCrossRefGoogle Scholar
  8. 8.
    van der Zee J, Gonzalez Gonzalez D, van Rhoon GC, van Dijk JD, van Putten WL, Hart AA (2000) Comparison of radiotherapy alone with radiotherapy plus hyperthermia in locally advanced pelvic tumours: a prospective, randomised, multicentre trial. Dutch Deep Hyperthermia Group. Lancet 355:1119–1125PubMedCrossRefGoogle Scholar
  9. 9.
    Vernon CC, Hand JW, Field SB et al (1996) Radiotherapy with or without hyperthermia in the treatment of superficial localized breast cancer: results from five randomized controlled trials. International Collaborative Hyperthermia Group. Int J Radiat Oncol Biol Phys 35:731–744PubMedCrossRefGoogle Scholar
  10. 10.
    Urano M, Kuroda M, Nishimura Y (1999) For the clinical application of thermochemotherapy given at mild temperatures. Int J Hyperthermia 15:79–107PubMedCrossRefGoogle Scholar
  11. 11.
    Dahl O, Mella O (2002) Referee: hyperthermia alone or combined with cisplatin in addition to radiotherapy for advanced uterine cervical cancer. Int J Hyperthermia 18:25–30PubMedCrossRefGoogle Scholar
  12. 12.
    Prosnitz L, Jones E (2002) Counterpoint: test the value of hyperthermia in patients with carcinoma of the cervix being treated with concurrent chemotherapy and radiation. Int J Hyperthermia 18:13–18PubMedCrossRefGoogle Scholar
  13. 13.
    Szostak MJ, Kyprianou N (2000) Radiation-induced apoptosis: predictive and therapeutic significance in radiotherapy of prostate cancer (review). Oncol Rep 7:699–706PubMedGoogle Scholar
  14. 14.
    Garzotto M, Haimovitz-Friedman A, Liao WC et al (1999) Reversal of radiation resistance in LNCaP cells by targeting apoptosis through ceramide synthase. Cancer Res 59:5194–5201PubMedGoogle Scholar
  15. 15.
    Lin X, Zhang F, Bradbury CM et al (2003) 2-Deoxy-D-glucose-induced cytotoxicity and radiosensitization in tumor cells is mediated via disruptions in thiol metabolism. Cancer Res 63:3413–3417PubMedGoogle Scholar
  16. 16.
    Belka C, Jendrossek V, Pruschy M, Vink S, Verheij M, Budach W (2004) Apoptosis-modulating agents in combination with radiotherapy-current status and outlook. Int J Radiat Oncol Biol Phys 58:542–554PubMedCrossRefGoogle Scholar
  17. 17.
    Salganik RI (2001) The benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population. J Am Coll Nutr 20:464S-472S; discussion 473S–475SPubMedGoogle Scholar
  18. 18.
    Shackelford RE, Kaufmann WK, Paules RS (2000) Oxidative stress and cell cycle checkpoint function. Free Radic Biol Med 28:1387–1404PubMedCrossRefGoogle Scholar
  19. 19.
    Davidson JF, Whyte B, Bissinger PH, Schiestl RH (1996) Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 93:5116–5121PubMedCrossRefGoogle Scholar
  20. 20.
    Flanagan SW, Moseley PL, Buettner GR (1998) Increased flux of free radicals in cells subjected to hyperthermia: detection by electron paramagnetic resonance spin trapping. FEBS Lett 431:285–286PubMedCrossRefGoogle Scholar
  21. 21.
    Frank J, Kelleher DK, Pompella A, Thews O, Biesalski HK, Vaupel P (1998) Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase. Cancer Res 58:2693–2698PubMedGoogle Scholar
  22. 22.
    Yoshikawa T, Kokura S, Tainaka K et al (1993) The role of active oxygen species and lipid peroxidation in the antitumor effect of hyperthermia. Cancer Res 53:2326–2329PubMedGoogle Scholar
  23. 23.
    Arai Y, Kondo T, Tanabe K et al (2002) Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells. J Biol Chem 277:18986–18993PubMedCrossRefGoogle Scholar
  24. 24.
    Cui ZG, Kondo T, Matsumoto H (2006) Enhancement of apoptosis by nitric oxide released from alpha-phenyl-tert-butyl nitrone under hyperthermic conditions. J Cell Physiol 206:468–476PubMedCrossRefGoogle Scholar
  25. 25.
    Li FJ, Kondo T, Zhao QL et al (2001) 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 35:281–299PubMedCrossRefGoogle Scholar
  26. 26.
    Yuki H, Kondo T, Zhao QL et al (2003) A free radical initiator, 2,2′-azobis (2-aminopropane) dihydrochloride enhances hyperthermia-induced apoptosis in human uterine cervical cancer cell lines. Free Radic Res 37:631–643PubMedCrossRefGoogle Scholar
  27. 27.
    Zhao QL, Fujiwara Y, Kondo T (2006) Mechanism of cell death induction by nitroxide and hyperthermia. Free Radic Biol Med 40:1131–1143PubMedCrossRefGoogle Scholar
  28. 28.
    Wada S, Cui ZG, Kondo T et al (2005) A hydrogen peroxide-generating agent, 6-formylpterin, enhances heat-induced apoptosis. Int J Hyperthermia 21:231–246PubMedGoogle Scholar
  29. 29.
    Cui ZG, Kondo T, Ogawa R et al (2004) Enhancement of radiation-induced apoptosis by 6-formylpterin. Free Radic Res 38:363–373PubMedCrossRefGoogle Scholar
  30. 30.
    Sellins KS, Cohen JJ (1987) Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J Immunol 139:3199–3206PubMedGoogle Scholar
  31. 31.
    Hopcia KL, McCarey YL, Sylvester FC, Held KD (1996) Radiation-induced apoptosis in HL60 cells: oxygen effect, relationship between apoptosis and loss of clonogenicity, and dependence of time to apoptosis on radiation dose. Radiat Res 145:315–323PubMedCrossRefGoogle Scholar
  32. 32.
    Zhao QL, Kondo T, Noda A, Fujiwara Y (1999) Mitochondrial and intracellular free-calcium regulation of radiation-induced apoptosis in human leukemic cells. Int J Radiat Biol 75:493–504PubMedCrossRefGoogle Scholar
  33. 33.
    van Heerde WL, de Groot PG, Reutelingsperger CP (1995) The complexity of the phospholipid binding protein Annexin V. Thromb Haemost 73:172–179PubMedGoogle Scholar
  34. 34.
    Datta R, Kojima H, Yoshida K, Kufe D (1997) Caspase-3-mediated cleavage of protein kinase C theta in induction of apoptosis. J Biol Chem 272:20317–20320PubMedCrossRefGoogle Scholar
  35. 35.
    Gorman A, McGowan A, Cotter TG (1997) Role of peroxide and superoxide anion during tumour cell apoptosis. FEBS Lett 404:27–33PubMedCrossRefGoogle Scholar
  36. 36.
    Royall JA, Ischiropoulos H (1993) Evaluation of 2′,7′-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 302:348–355PubMedCrossRefGoogle Scholar
  37. 37.
    Tsujimoto Y, Shimizu S (2000) Bcl-2 family: life-or-death switch. FEBS Lett 466:6–10PubMedCrossRefGoogle Scholar
  38. 38.
    Katschinski DM, Boos K, Schindler SG, Fandrey J (2000) Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis. J Biol Chem 275:21094–21098PubMedCrossRefGoogle Scholar
  39. 39.
    Skibba JL, Quebbeman EJ, Kalbfleisch JH (1986) Nitrogen metabolism and lipid peroxidation during hyperthermic perfusion of human livers with cancer. Cancer Res 46:6000–6003PubMedGoogle Scholar
  40. 40.
    Green DR (2000) Apoptotic pathways: paper wraps stone blunts scissors. Cell 102:1–4PubMedCrossRefGoogle Scholar
  41. 41.
    Wang X (2001) The expanding role of mitochondria in apoptosis. Genes Dev 15:2922–2933PubMedGoogle Scholar
  42. 42.
    Medema JP, Scaffidi C, Kischkel FC et al (1997) FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). Embo J 16:2794–2804PubMedCrossRefGoogle Scholar
  43. 43.
    Nagata S (1997) Apoptosis by death factor. Cell 88:355–365PubMedCrossRefGoogle Scholar
  44. 44.
    Scaffidi C, Medema JP, Krammer PH, Peter ME (1997) FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b. J Biol Chem 272:26953–26958PubMedCrossRefGoogle Scholar
  45. 45.
    Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R (2001) A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell 8:613–621PubMedCrossRefGoogle Scholar
  46. 46.
    Adams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326PubMedCrossRefGoogle Scholar
  47. 47.
    Huang DC, Tschopp J, Strasser A (2000) Bcl-2 does not inhibit cell death induced by the physiological Fas ligand: implications for the existence of type I and type II cells. Cell Death Differ 7:754–755PubMedCrossRefGoogle Scholar
  48. 48.
    Eskes R, Desagher S, Antonsson B, Martinou JC (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 20:929–935PubMedCrossRefGoogle Scholar
  49. 49.
    Wei MC, Lindsten T, Mootha VK et al (2000) tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14:2060–2071PubMedGoogle Scholar
  50. 50.
    Nitobe J, Yamaguchi S, Okuyama M et al (2003) Reactive oxygen species regulate FLICE inhibitory protein (FLIP) and susceptibility to Fas-mediated apoptosis in cardiac myocytes. Cardiovasc Res 57:119–128PubMedCrossRefGoogle Scholar
  51. 51.
    Strasser A, Newton K (1999) FADD/MORT1, a signal transducer that can promote cell death or cell growth. Int J Biochem Cell Biol 31:533–537PubMedCrossRefGoogle Scholar
  52. 52.
    Yin XM (2000) Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways. Cell Res 10:161–167PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Da-Yong Yu
    • 1
  • Yuji Matsuya
    • 2
  • Qing-Li Zhao
    • 1
  • Kanwal Ahmed
    • 1
  • Zheng-Li Wei
    • 1
  • Hideo Nemoto
    • 2
  • Takashi Kondo
    • 1
    Email author
  1. 1.Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  2. 2.Laboratory of Medicinal Chemistry, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan

Personalised recommendations