Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
권호 표지
DOI QR코드

DOI QR Code

Shikonin Induces Apoptotic Cell Death via Regulation of p53 and Nrf2 in AGS Human Stomach Carcinoma Cells

  • Ko, Hyeonseok (Laboratory of Molecular Oncology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine) ;
  • Kim, Sun-Joong (College of Life Sciences & Biotechnology, College of Medicine, Korea University) ;
  • Shim, So Hee (Department of Microbiology, College of Medicine, Korea University) ;
  • Chang, HyoIhl (College of Life Sciences & Biotechnology, College of Medicine, Korea University) ;
  • Ha, Chang Hoon (Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center)
  • Received : 2016.01.12
  • Accepted : 2016.04.05
  • Published : 2016.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

Shikonin, which derives from Lithospermum erythrorhizon, has been traditionally used against a variety of diseases, including cancer, in Eastern Asia. Here we determined that shikonin inhibits proliferation of gastric cancer cells by inducing apoptosis. Shikonin's biological activity was validated by observing cell viability, caspase 3 activity, reactive oxygen species (ROS) generation, and apoptotic marker expressions in AGS stomach cancer cells. The concentration range of shikonin was 35-250 nM with the incubation time of 6 h. Protein levels of Nrf2 and p53 were evaluated by western blotting and confirmed by real-time PCR. Our results revealed that shikonin induced the generation of ROS as well as caspase 3-dependent apoptosis. c-Jun-N-terminal kinases (JNK) activity was significantly elevated in shikonin-treated cells, thereby linking JNK to apoptosis. Furthermore, our results revealed that shikonin induced p53 expression but repressed Nrf2 expression. Moreover, our results suggested that there may be a co-regulation between p53 and Nrf2, in which transfection with siNrf2 induced the p53 expression. We demonstrated for the first time that shikonin activated cell apoptosis in AGS cells via caspase 3- and JNK-dependent pathways, as well as through the p53-Nrf2 mediated signal pathway. Our study validates in partly the contribution of shikonin as a new therapeutic approaches/agent for cancer chemotherapy.

Keywords

References

  1. Akhdar, H., Loyer, P., Rauch, C., Corlu, A., Guillouzo, A. and Morel, F. (2009) Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells. Eur. J. Cancer 45, 2219-2227. https://doi.org/10.1016/j.ejca.2009.05.017
  2. Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M. and Cook, J. L. (1999) Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J. Biol. Chem. 274, 26071-26078. https://doi.org/10.1074/jbc.274.37.26071
  3. Chen, X., Yang, L., Oppenheim, J. J. and Howard, M. Z. (2002) Cellular pharmacology studies of shikonin derivatives. Phytother. Res. 16, 199-209. https://doi.org/10.1002/ptr.1100
  4. Chu, F. F., Esworthy, R. S. and Doroshow, J. H. (2004) Role of Se-dependent glutathione peroxidases in gastrointestinal inflammation and cancer. Free Radic. Biol. Med. 36, 1481-1495. https://doi.org/10.1016/j.freeradbiomed.2004.04.010
  5. Devasagayam, T. P., Tilak, J. C., Boloor, K. K., Sane, K. S., Ghaskadbi, S. S. and Lele, R. D. (2004) Free radicals and antioxidants in human health: current status and future prospects. J. Assoc. Physicians India 52, 794-804.
  6. Faraonio, R., Vergara, P., Di Marzo, D., Pierantoni, M. G., Napolitano, M., Russo, T. and Cimino, F. (2006) p53 suppresses the Nrf2-dependent transcription of antioxidant response genes. J. Biol. Chem. 281, 39776-39784. https://doi.org/10.1074/jbc.M605707200
  7. Filomeni, G., Aquilano, K., Rotilio, G. and Ciriolo, M. R. (2005a) Glutathione-related systems and modulation of extracellular signalregulated kinases are involved in the resistance of AGS adenocarcinoma gastric cells to diallyl disulfide-induced apoptosis. Cancer Res. 65, 11735-11742. https://doi.org/10.1158/0008-5472.CAN-05-3067
  8. Filomeni, G., Rotilio, G. and Ciriolo, M. R. (2005b) Disulfide relays and phosphorylative cascades: partners in redox-mediated signaling pathways. Cell Death Differ. 12, 1555-1563. https://doi.org/10.1038/sj.cdd.4401754
  9. Fujii, N., Yamashita, Y., Arima, Y., Nagashima, M. and Nakano, H. (1992) Induction of topoisomerase II-mediated DNA cleavage by the plant naphthoquinones plumbagin and shikonin. Antimicrob. Agents Chemother. 36, 2589-2594. https://doi.org/10.1128/AAC.36.12.2589
  10. Guo, X. P., Zhang, X. Y. and Zhang, S. D. (1991) [Clinical trial on the effects of shikonin mixture on later stage lung cancer]. Zhong Xi Yi Jie He Za Zhi, 11, 598-599, 580.
  11. Hsu, P. C., Huang, Y. T., Tsai, M. L., Wang, Y. J., Lin, J. K. and Pan, M. H. (2004) Induction of apoptosis by shikonin through coordinative modulation of the Bcl-2 family, p27, and p53, release of cytochrome c, and sequential activation of caspases in human colorectal carcinoma cells. J. Agric. Food Chem. 52, 6330-6337. https://doi.org/10.1021/jf0495993
  12. Jones, D. V., Jr., Winn, R. J., Brown, B. W., Levy, L. B., Pugh, R. P., Wade, J. L., 3rd, Gross, H. M., Pendergrass, K. B., Levin, B. and Abbruzzese, J. L. (1995) Randomized phase III study of 5-fluorouracil plus high dose folinic acid versus 5-fluorouracil plus folinic acid plus methyl-lomustine for patients with advanced colorectal cancer. Cancer 76, 1709-1714. https://doi.org/10.1002/1097-0142(19951115)76:10<1709::AID-CNCR2820761006>3.0.CO;2-5
  13. Kim, S. J., Lee, H. J., Kim, B. S., Lee, D., Lee, S. J., Yoo, S. H. and Chang, H. I. (2011a) Antiulcer activity of anthocyanins from Rubus coreanus via association with regulation of the activity of matrix metalloproteinase-2. J. Agric. Food Chem. 59, 11786-11793. https://doi.org/10.1021/jf104192a
  14. Kim, S. J., Park, Y. S., Paik, H. D. and Chang, H. I. (2011b) Effect of anthocyanins on expression of matrix metalloproteinase-2 in naproxen-induced gastric ulcers. Br. J. Nutr. 106, 1792-1801. https://doi.org/10.1017/S000711451100242X
  15. Kim, S. J., Kim, J. M., Shim, S. H. and Chang, H. I. (2014a) Shikonin induces cell cycle arrest in human gastric cancer (AGS) by early growth response 1 (Egr1)-mediated p21 gene expression. J. Ethnopharmacol. 151, 1064-1071. https://doi.org/10.1016/j.jep.2013.11.055
  16. Kim, Y. J., Choi, W. I., Jeon, B. N., Choi, K. C., Kim, K., Kim, T. J., Ham, J., Jang, H. J., Kang, K. S. and Ko, H. (2014b) Stereospecific effects of ginsenoside 20-Rg3 inhibits TGF-${\beta}1$-induced epithelialmesenchymal transition and suppresses lung cancer migration, invasion and anoikis resistance. Toxicology 322, 23-33. https://doi.org/10.1016/j.tox.2014.04.002
  17. Ko, H., Kim, S., Jin, C. H., Lee, E., Ham, S., Yook, J. I. and Kim, K. (2012) Protein kinase casein kinase 2-mediated upregulation of N-cadherin confers anoikis resistance on esophageal carcinoma cells. Mol. Cancer Res. 10, 1032-1038. https://doi.org/10.1158/1541-7786.MCR-12-0261
  18. Ko, H., So, Y., Jeon, H., Jeong, M. H., Choi, H. K., Ryu, S. H., Lee, S. W., Yoon, H. G. and Choi, K. C. (2013) TGF-${\beta}1$-induced epithelialmesenchymal transition and acetylation of Smad2 and Smad3 are negatively regulated by EGCG in human A549 lung cancer cells. Cancer Lett. 335, 205-213. https://doi.org/10.1016/j.canlet.2013.02.018
  19. Kwak, M. K., Wakabayashi, N., Itoh, K., Motohashi, H., Yamamoto, M. and Kensler, T. W. (2003) Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway. Identification of novel gene clusters for cell survival. J. Biol. Chem. 278, 8135-8145. https://doi.org/10.1074/jbc.M211898200
  20. Lee, H. and Lin, J. Y. (1988) Antimutagenic activity of extracts from anticancer drugs in Chinese medicine. Mutat. Res. 204, 229-234. https://doi.org/10.1016/0165-1218(88)90093-6
  21. Leung, P.-C. and Fong, H. H. S. (2007) Alternative treatment for cancer. World Scientific, New Jersey.
  22. Liu, C., Tseng, A. and Yang, S. (2005) Chinese herbal medicine: modern applications of traditional formulas. CRC Press, Florida.
  23. Macip, S., Igarashi, M., Berggren, P., Yu, J., Lee, S. W. and Aaronson, S. A. (2003) Influence of induced reactive oxygen species in p53-mediated cell fate decisions. Mol. Cell. Biol. 23, 8576-8585. https://doi.org/10.1128/MCB.23.23.8576-8585.2003
  24. Mao, X., Yu, C. R., Li, W. H. and Li, W. X. (2008) Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Ablpositive chronic myelogenous leukemia (CML) cells. Cell Res. 18, 879-888. https://doi.org/10.1038/cr.2008.86
  25. Matsuzawa, A. and Ichijo, H. (2005) Stress-responsive protein kinases in redox-regulated apoptosis signaling. Antioxid. Redox Signal. 7, 472-481. https://doi.org/10.1089/ars.2005.7.472
  26. Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W. and Vogelstein, B. (1997) A model for p53-induced apoptosis. Nature 389, 300-305. https://doi.org/10.1038/38525
  27. Seger, R. and Krebs, E. G. (1995) The MAPK signaling cascade. FASEB J. 9, 726-735. https://doi.org/10.1096/fasebj.9.9.7601337
  28. Shim, S. H., Park, M. S., Moon, S., Park, K. S., Song, J. W., Song, K. J. and Baek, L. J. (2011) Comparison of innate immune responses to pathogenic and putative non-pathogenic hantaviruses in vitro. Virus Res. 160, 367-373. https://doi.org/10.1016/j.virusres.2011.07.013
  29. Shin, D. H., Park, H. M., Jung, K. A., Choi, H. G., Kim, J. A., Kim, D. D., Kim, S. G., Kang, K. W., Ku, S. K., Kensler, T. W. and Kwak, M. K. (2010) The NRF2-heme oxygenase-1 system modulates cyclosporin A-induced epithelial-mesenchymal transition and renal fibrosis. Free Radic. Biol. Med. 48, 1051-1063. https://doi.org/10.1016/j.freeradbiomed.2010.01.021
  30. Simon, H. U., Haj-Yehia, A. and Levi-Schaffer, F. (2000) Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5, 415-418. https://doi.org/10.1023/A:1009616228304
  31. Wakabayashi, N., Slocum, S. L., Skoko, J. J., Shin, S. and Kensler, T. W. (2010) When NRF2 talks, who's listening? Antioxid. Redox Signal. 13, 1649-1663. https://doi.org/10.1089/ars.2010.3216
  32. Wu, Z., Wu, L., Li, L., Tashiro, S., Onodera, S. and Ikejima, T. (2004) p53-mediated cell cycle arrest and apoptosis induced by shikonin via a caspase-9-dependent mechanism in human malignant melanoma A375-S2 cells. J. Pharmacol. Sci, 94, 166-176. https://doi.org/10.1254/jphs.94.166

Cited by

  1. Dioscin suppresses TGF-β1-induced epithelial-mesenchymal transition and suppresses A549 lung cancer migration and invasion vol.27, pp.15, 2017, https://doi.org/10.1016/j.bmcl.2017.06.014
  2. Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway vol.60, 2017, https://doi.org/10.1016/j.procbio.2017.05.024
  3. Combined treatment with zingerone and its novel derivative synergistically inhibits TGF-β1 induced epithelial-mesenchymal transition, migration and invasion of human hepatocellular carcinoma cells vol.27, pp.4, 2017, https://doi.org/10.1016/j.bmcl.2016.12.042
  4. Shikonin induces mitochondria-mediated apoptosis and attenuates epithelial-mesenchymal transition in cisplatin-resistant human ovarian cancer cells vol.15, pp.4, 2018, https://doi.org/10.3892/ol.2018.8065
  5. Efficacy of Shikonin against Esophageal Cancer Cells and its possible mechanisms in vitro and in vivo vol.9, pp.1, 2016, https://doi.org/10.7150/jca.21224
  6. Advanced Drug Delivery Nanosystems for Shikonin: A Calorimetric and Electron Paramagnetic Resonance Study vol.34, pp.32, 2016, https://doi.org/10.1021/acs.langmuir.8b00751
  7. MS-5, a Naphthalene Derivative, Induces the Apoptosis of an Ovarian Cancer Cell CAOV-3 by Interfering with the Reactive Oxygen Species Generation vol.27, pp.1, 2019, https://doi.org/10.4062/biomolther.2018.020
  8. Combination of shikonin with paclitaxel overcomes multidrug resistance in human ovarian carcinoma cells in a P-gp-independent manner through enhanced ROS generation vol.14, pp.None, 2019, https://doi.org/10.1186/s13020-019-0231-3
  9. RETRACTED ARTICLE: Shikonin inhibits proliferation, migration, invasion and promotes apoptosis in NCI-N87 cells via inhibition of PI3K/AKT signal pathway vol.47, pp.1, 2016, https://doi.org/10.1080/21691401.2019.1632870
  10. A New TGF-β1 Inhibitor, CTI-82, Antagonizes Epithelial–Mesenchymal Transition through Inhibition of Phospho-SMAD2/3 and Phospho-ERK vol.9, pp.7, 2016, https://doi.org/10.3390/biology9070143
  11. Molecular mechanism of shikonin inhibiting tumor growth and potential application in cancer treatment vol.10, pp.6, 2016, https://doi.org/10.1093/toxres/tfab107