Persimmon Leaf Flavonols Enhance the Anti-Cancer Effect of Heavy Ion Radiotherapy on Murine Xenograft Tumors


The cell cycle checkpoint system play a pivotal role in the cellular DNA damage response, and the discovery of checkpoint inhibitors is expected to sensitize current cancer therapies. Checkpoint signaling cascades are critically modulated by ATM (ataxia telangiectasia-mutated) and its related molecules. Generally, ATM primarily responds to ionizing irradiation-induced DNA double-strand breaks. Heavy ions from an accelerated carbon ion beam have been used to cure cancer because they are more effective than ionizing irradiation such as X-ray and γ-radiation in terms of biological damage. In a previous study, we demonstrated that a persimmon leaf flavonol (PLF) promoted the cytotoxic effect of chemotherapeutic agents on cancer cells through inhibition of checkpoint activities, especially in the ATM dependent pathway. The present study investigated whether PLF inhibits checkpoint activity during the DNA damage response induced by heavy ion irradiation. Treatment with PLF significantly increased the cytotoxicity of heavy ion irradiation in A549 adenocarcinoma cells. The phosphorylation of checkpoint proteins such as p53, SMC1, and Chk1 was increased by heavy ions. PLF reduced the phosphorylation of checkpoint proteins. Pre-treatment with PLF significantly prevented the decrease of mitotic cells in heavy ion-exposed cells. We further evaluated tumor volume in SCID mice inoculated with human lung adenocarcinoma A549 cells. The combination treatment of PLF and heavy ion resulted in a decrease of tumor volume compared with controls, although PLF itself did not exhibit any effect. These results indicate that PLF inhibits tumor growth through modulation of the DNA damage response. PLF may be useful for clinical application in combination with heavy ion radiotherapy.

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K. Kawakami, H. Nishida, N. Tatewaki, K. Eguchi-Kasai, K. Anzai, T. Eitsuka, T. Konishi and M. Hirayama, "Persimmon Leaf Flavonols Enhance the Anti-Cancer Effect of Heavy Ion Radiotherapy on Murine Xenograft Tumors," Journal of Cancer Therapy, Vol. 4 No. 7, 2013, pp. 1150-1157. doi: 10.4236/jct.2013.47133.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] P. Todd, J. C. Wood, J. T. Walker and S. J. Weiss, “Lethal, Potentially Lethal, and Nonlethal Damage Induction by Heavy Ions in Cultured Human Cells,” Radiation Research Supplement, Vol. 8, 1985, pp. S5-S12. doi:10.2307/3576625
[2] N. Hamada, T. Imaoka, S. Masunaga, T. Ogata, R. Okayasu, A. Takahashi, T. A. Kato, Y. Kobayashi, T. Ohnishi, K. Ono, Y. Shimada and T. Teshima, “Recent Advances in the Biology of Heavy-Ion Cancer Therapy,” Journal of Radiation Research, Vol. 51, No. 4, 2010, pp. 365-383. doi:10.1269/jrr.09137
[3] T. Ohno, “Particle Radiotherapy with Carbon Ion Beams,” EPMA Journal, Vol. 4, No. 1, 2013, p. 9. doi:10.1186/1878-5085-4-9
[4] G. M. Ross, “Induction of Cell Death by Radiotherapy,” Endocrine-Related Cancer, Vol. 6, No. 1, 1999, pp. 41-44. doi:10.1677/erc.0.0060041
[5] Y. Shiloh, “ATM and ATR: Networking Cellular Responses to DNA Damage,” Current Opinion in Genetics & Development, Vol. 11, No. 1, 2001, pp. 71-77. doi:10.1016/S0959-437X(00)00159-3
[6] R. T. Abraham, “Cell Cycle Checkpoint Signaling through the ATM and ATR Kinases,” Genes & Development, Vol. 15, No. 17, 2001, pp. 2177-2196. doi:10.1101/gad.914401
[7] S. Matsuoka, B. A. Ballif, A. Smogorzewska, E. R. McDonald, K. E. Hurov, J. Luo, C. E. Bakalarski, Z. Zhao, N. Solimini, Y. Lerenthal, Y. Shiloh, S. P. Gygi and S. J. Elledge, “ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage,” Science, Vol. 316, No. 5828, 2007, pp. 1160-1166. doi:10.1126/science.1140321
[8] S. N. Powell, J. S. DeFrank, P. Connell, M. Eogan, F. Preffer, D. Dombkowski, W. Tang and S. Friend, “Differential Sensitivity of p53(-) and p53(+) Cells to Caffeine-Induced Radiosensitization and Override of G2 Delay,” Cancer Research, Vol. 55, No. 8, 1995, pp. 1643-1648.
[9] H. Nishida, N. Tatewaki, Y. Nakajima, T. Magara, K. M. Ko, Y. Hamamori and T. Konishi, “Inhibition of ATR Protein Kinase Activity by Schisandrin B in DNA Damage Response,” Nucleic Acids Research, Vol. 37, No. 17, 2009, pp. 5678-5689. doi:10.1093/nar/gkp593
[10] K. Kawakami, H. Nishida, N. Tatewaki, Y. Nakajima, T. Konishi and M. Hirayama, “Persimmon Leaf Extract Inhibits the ATM Activity during DNA Damage Response Induced by Doxorubicin in A549 Lung Adenocarcinoma Cells,” Bioscience, Biotechnology, and Biochemistry, Vol. 75, No. 4, 2011, pp. 650-655. doi:10.1271/bbb.100738
[11] S. Sakanaka, Y. Tachibana and Y. Okada, “Preparation and Antioxidant Properties of Extracts of Japanese Persimmon Leaf Tea (Kakinoha-Cha),” Food Chemistry, Vol. 89, No. 4, 2005, pp. 569-575. doi:10.1016/j.foodchem.2004.03.013
[12] W. Bei, W. Peng, Y. Ma and A. Xu, “Flavonoids from the Leaves of Diospyros kaki Reduce Hydrogen PeroxideInduced Injury of NG108-15 Cells,” Life Science, Vol. 76, No. 17, 2005, pp. 1975-1988. doi:10.1016/j.lfs.2004.09.031
[13] L. Sun, J. Zhang, X. Lu, L. Zhang and Y. Zhang, “Evaluation to the Antioxidant Activity of Total Flavonoids Extract from Persimmon (Diospyros kaki L.) Leaves,” Food and Chemical Toxicology, Vol. 49, No. 10, 2011, pp. 2689-2696. doi:10.1016/j.fct.2011.07.042
[14] G. Chen, J. Xue, S. X. Xu and R. Q. Zhang, “Chemical Constituents of the Leaves of Diospyros kaki and Their Cytotoxic Effects,” Journal of Asian Natural Products Research, Vol. 9, No. 3-5, 2007, pp. 347-353. doi:10.1080/10286020600727731
[15] P. Khanal, W. K. Oh, P. T. Thuong, S. D. Cho and H. S. Choi, “24-Hydroxyursolic Acid from the Leaves of the Diospyros kaki (Persimmon) Induces Apoptosis by Activation of AMP-Activated Protein Kinase,” Planta Medica, Vol. 76, No. 7, 2010, pp. 689-693. doi:10.1055/s-0029-1240678
[16] K. Kameda, T. Takaku, H. Okuda, Y. Kimura, T. Okuda, T. Hatano, I. Agata and S. Arichi, “Inhibitory Effects of Various Flavonoids Isolated from Leaves of Persimmon on Angiotensin-Converting Enzyme Activity,” Journal of Natural Products, Vol. 50, No. 4, 1987, pp. 680-683. doi:10.1021/np50052a017
[17] K. Kawakami, S. Aketa, H. Sakai, Y. Watanabe, H. Nishida and M. Hirayama, “Antihypertensive and Vasorelaxant Effects of Water-Soluble Proanthocyanidins from Persimmon Leaf Tea in Spontaneously Hypertensive Rats,” Bioscience, Biotechnology, and Biochemistry, Vol. 75, No. 8, 2011, pp. 1435-1439. doi:10.1271/bbb.100926
[18] M. Kotani, M. Matsumoto, A. Fujita, S. Higa, W. Wang, M. Suemura, T. Kishimoto and T. Tanaka, “Persimmon Leaf Extract and Astragalin Inhibit Development of Dermatitis and IgE Elevation in NC/Nga Mice,” Journal of Allergy and Clinical Immunology, Vol. 106, No. 1, 2000, pp. 159-166. doi:10.1067/mai.2000.107194
[19] M. Matsumoto, M. Kotani, A. Fujita, S. Higa, T. Kishimoto, M. Suemura and T. Tanaka, “Oral Administration of Persimmon Leaf Extract Ameliorates Skin Symptoms and Transepidermal Water Loss in Atopic Dermatitis Model Mice, NC/Nga,” British Journal of Dermatology, Vol. 146, No. 2, 2002, pp. 221-227. doi:10.1046/j.1365-2133.2002.04557.x
[20] K. Kawakami, S. Aketa, M. Nakanami, S. Iizuka and M. Hirayama, “Major Water-Soluble Polyphenols, Proanthocyanidins, in Leaves of Persimmon (Diospyros kaki) and Their Alpha-Amylase Inhibitory Activity,” Bioscience, Biotechnology, and Biochemistry, Vol. 74, No. 7, 2010, pp. 1380-1385. doi:10.1271/bbb.100056
[21] U. J. Jung, Y. B. Park, S. R. Kim and M. S. Choi, “Supplementation of Persimmon Leaf Ameliorates Hyperglycemia, Dyslipidemia and Hepatic Fat Accumulation in Type 2 Diabetic Mice,” PLoS One, Vol. 7, No. 11, 2012, Article ID: e49030. doi:10.1371/journal.pone.0049030
[22] K. Kawakami, Y. Shibukura, T. Kanno, T. Furuki, S. Aketa and M. Hirayama, “Identification of 2''-Galloylated Flavonol 3-O-Glycosides Accumulating in Developing Leaves of Persimmon,” Phytochemical Analysis, Vol. 22, No. 5, 2011, pp. 403-410. doi:10.1002/pca.1295
[23] S. Kitajima, H. Nakamura, M. Adachi, K. Ijichi, Y. Yasui, N. Saito, M. Suzuki, K. Kurita and K. Ishizaki, “AT Cells Show Dissimilar Hypersensitivity to Heavy-Ion and XRays Irradiation,” Journal of Radiation Research, Vol. 51, No. 3, 2010, pp. 251-255. doi:10.1269/jrr.09069
[24] S. Ghosh, H. Narang, A. Sarma and M. Krishna, “DNA Damage Response Signaling in Lung Adenocarcinoma A549 Cells Following Gamma and Carbon Beam Irradiation,” Mutation Research, Vol. 716, No. 1-2, 2011, pp. 10-19. doi:10.1016/j.mrfmmm.2011.07.015
[25] L, Xue, D. Yu, Y. Furusawa, J. Cao, R. Okayasu and S. Fan, “ATM-Dependent Hyper-Radiosensitivity in Mammalian Cells Irradiated by Heavy Ions,” International Journal of Radiation Oncology, Biology, Physics, Vol. 75, No. 1, 2009, pp. 235-243.
[26] D. T. Goodhead, “Initial Events in the Cellular Effects of Ionizing Radiations: Clustered Damage in DNA,” International Journal of Radiation Biology, Vol. 65, No. 1, 1994, pp. 7-17. doi:10.1080/09553009414550021
[27] M. Hada and A. G. Georgakilas, “Formation of Clustered DNA Damage after High-LET Irradiation: A Review,” Journal of Radiation Research, Vol. 49, No. 3, 2008, pp. 203-210. doi:10.1269/jrr.07123
[28] A. J. Levine, “p53, the Cellular Gatekeeper for Growth and Division,” Cell, Vol. 88, No. 3, 1997, pp. 323-331. doi:10.1016/S0092-8674(00)81871-1
[29] Y. Wang, J. Li, R. N. Booher, A. Kraker, T. Lawrence, W. R. Leopold and Y. Sun, “Radiosensitization of p53 Mutant Cells by PD0166285, a Novel G(2) Checkpoint Abrogator,” Cancer Research, Vol. 61, No. 22, 2001, pp. 8211-8217.
[30] T. Kawabe, “G2 Checkpoint Abrogators as Anticancer Drugs,” Molecular Cancer Therapeutics, Vol. 3, No. 4, 2004, pp. 513-519.
[31] D. P. Liu, H. Song and Y. Xu, “A Common Gain of Function of p53 Cancer Mutants in Inducing Genetic Instability,” Oncogene, Vol. 29, No. 7, 2010, pp. 949-956. doi:10.1038/onc.2009.376
[32] T. Hatano, T. Yasuhara, R. Yoshihara, Ikegami, Y. M. Matsuda, K. Yazaki, I. Agata, S. Nishibe, T. Noro and M. Yoshizaki, “Inhibitory Effects of Galloylated Flavonoids on Xanthine Oxidase,” Planta Medica, Vol. 57, No. 1, 1991, pp. 83-84. doi:10.1055/s-2006-960028
[33] M. J. Ahn, C. Y. Kim, J. S. Lee, T. G. Kim, S. H. Kim, C. K. Lee, B. B. Lee, C. G. Shin, H. Huh and J. Kim, “Inhibition of HIV-1 Integrase by Galloyl Glucoses from Terminalia Chebula and Flavonol Glycoside Gallates from Euphorbia pekinensis,” Planta Medica, Vol. 68, No. 5, 2002, pp. 457-459. doi:10.1055/s-2002-32070
[34] H. Y. Jo, Y. Kim, S. Y. Nam, Lee, B. J. Y. B. Kim, Y. W. Yun and B. Ahn, “The Inhibitory Effect of Quercitrin Gallate on iNOS Expression Induced by Lipopolysaccharide in Balb/c Mice,” Journal of Veterinary Science, Vol. 9, No. 3, 2008, pp. 267-272. doi:10.4142/jvs.2008.9.3.267
[35] A. J. Chalmers, E. M. Ruff, C. Martindale, N. Lovegrove and S. C. Short, “Cytotoxic Effects of Temozolomide and Radiation Are Additiveand Schedule-Dependent,” International Journal of Radiation Oncology, Biology, Physics, Vol. 75, No. 5, 2009, pp. 1511-1519. doi:10.1016/j.ijrobp.2009.07.1703
[36] V. Gavrilov, Y. Leibovich, S. Ariad, K. Lavrenkov and S. Shany, “A Combined Pretreatment of 1,25-Dihydroxyvitamin D3 and Sodium Valproate Enhances the Damaging Effect of Ionizing Radiation on Prostate Cancer Cells,” Journal of Steroid Biochemistry and Molecular Biology, Vol. 121, No. 1-2, 2010, pp. 391-394. doi:10.1016/j.jsbmb.2010.03.004
[37] M. A. Morgan, L. A. Parsels, L. Zhao, Parsels, J. D. M. A. Davis, M. C. Hassan, S. Arumugarajah, L. HylanderGans, D. Morosini, D. M. Simeone, C. E. Canman, D. P. Normolle, S. D. Zabludoff, J. Maybaum and T. S. Lawrence, “Mechanism of Radiosensitization by the Chk1/2 Inhibitor AZD7762 Involves Abrogation of the G2 Checkpoint and Inhibition of Homologous Recombinational DNA Repair,” Cancer Research, Vol. 70, No. 12, 2010, pp. 4972-4981. doi:10.1158/0008-5472.CAN-09-3573
[38] S. E. Golding, E. Rosenberg, N. Valerie, I. Hussaini, M. Frigerio, X. F. Cockcroft, W. Y. Chong, M. Hummersone, L. Rigoreau, K. A. Menear, M. J. O’Connor, L. F. Povirk, T. van Meter and K. Valerie, “Improved ATM Kinase Inhibitor KU-60019 Radiosensitizes Glioma Cells, Compromises Insulin, AKT and ERK Prosurvival Signaling, and Inhibits Migration and Invasion,” Molecular Cancer Therapeutics, Vol. 8, No. 10, 2009, pp. 2894-2902. doi:10.1158/1535-7163.MCT-09-0519
[39] D. J. Newman and G. M. Cragg, “Natural Products as Sources of New Drugs over the Last 25 Years,” Journal of Natural Products, Vol. 70, No. 3, 2007, pp. 461-477. doi:10.1021/np068054v

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