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Auranofin Enhances Sulforaphane-Mediated Apoptosis in Hepatocellular Carcinoma Hep3B Cells through Inactivation of the PI3K/Akt Signaling Pathway

  • Hwangbo, Hyun (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Kim, So Young (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Lee, Hyesook (Anti-Aging Research Center, Dong-eui University) ;
  • Park, Shin-Hyung (Department of Pathology, Dong-eui University College of Korean Medicine) ;
  • Hong, Su Hyun (Department of Biochemistry, Dong-eui University College of Korean Medicine) ;
  • Park, Cheol (Division of Basic Sciences, College of Liberal Studies, Dong-eui University) ;
  • Kim, Gi-Young (Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University) ;
  • Leem, Sun-Hee (Department of Biological Science, College of Natural Sciences, Dong-A University) ;
  • Hyun, Jin Won (Jeju National University School of Medicine and Jeju Research Center for Natural Medicine) ;
  • Cheong, Jaehun (Department of Molecular Biology, College of Natural Sciences, Pusan National University) ;
  • Choi, Yung Hyun (Anti-Aging Research Center, Dong-eui University)
  • Received : 2020.07.09
  • Accepted : 2020.07.22
  • Published : 2020.09.01

Abstract

The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.

Keywords

References

  1. Arnér, E. S. and Holmgren, A. (2000) Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267, 6102-6109. https://doi.org/10.1046/j.1432-1327.2000.01701.x
  2. Baffy, G., Brunt, E. M. and Caldwell, S. H. (2012) Hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace. J. Hepatol. 56, 1384-1391. https://doi.org/10.1016/j.jhep.2011.10.027
  3. Becker, K., Gromer, S., Schirmer, R. H. and Muller, S. (2000) Thioredoxin reductase as a pathophysiological factor and drug target. Eur. J. Biochem. 267, 6118-6125. https://doi.org/10.1046/j.1432-1327.2000.01703.x
  4. Cheng, S., Zhang, X., Feng, Q., Chen, J., Shen, L., Yu, P., Yang, L., Chen, D., Zhang, H., Sun, W. and Chen, X. (2019) Astragaloside IV exerts angiogenesis and cardioprotection after myocardial infarction via regulating PTEN/PI3K/Akt signaling pathway. Life Sci. 227, 82-93. https://doi.org/10.1016/j.lfs.2019.04.040
  5. Cheng, Y. and Qi, Y. (2017) Current progresses in metal-based anticancer complexes as mammalian TrxR inhibitors. Anticancer Agents Med. Chem. 17, 1046-1069.
  6. Conklin, K. A. (2000) Dietary antioxidants during cancer chemotherapy: impact on chemotherapeutic effectiveness and development of side effects. Nutr. Cancer 37, 1-18. https://doi.org/10.1207/S15327914NC3701_1
  7. Cox, A. G., Brown, K. K., Arner, E. S. and Hampton, M. B. (2008) The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves peroxiredoxin 3 oxidation. Biochem. Pharmacol. 76, 1097-1109. https://doi.org/10.1016/j.bcp.2008.08.021
  8. Emens, L. A. and Middleton, G. (2015) The interplay of immunotherapy and chemotherapy: harnessing potential synergies. Cancer Immunol. Res. 3, 436-443. https://doi.org/10.1158/2326-6066.CIR-15-0064
  9. Fang, J. and Holmgren, A. (2006) Inhibition of thioredoxin and thioredoxin reductase by 4-hydroxy-2-nonenal in vitro and in vivo. J. Am. Chem. Soc. 128, 1879-1885. https://doi.org/10.1021/ja057358l
  10. Ferlay, J., Ervik, M., Lam, F., Colombet, M., Mery, L., Piñeros, M., Znaor, A., Soerjomataram, I. and Bray, F. (2018) Global Cancer Observatory: Cancer Today. International Agency for Research on Cancer, Lyon. Available from: https://gco.iarc.fr/today/ [accessed 2019 Mar 25].
  11. Gamet-Payrastre, L., Li, P., Lumeau, S., Cassar, G., Dupont, M. A., Chevolleau, S., Gasc, N., Tulliez, J. and Terce, F. (2000) Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res. 60, 1426-1433.
  12. Gerl, R. and Vaux, D. L. (2005) Apoptosis in the development and treatment of cancer. Carcinogenesis 26, 263-270. https://doi.org/10.1093/carcin/bgh283
  13. Green, D. R. and Llambi, F. (2015) Cell death signaling. Cold Spring Harb. Perspect. Biol. 7, a006080.
  14. Hasan, M. M., Islam, M. S., Hoque, K. M. F., Haque, A. and Reza, M. A. (2019) Effect of Citrus macroptera fruit pulp juice on alteration of caspase pathway rendering anti-proliferative activity against Ehrlich's ascites carcinoma in mice. Toxicol. Res. 35, 271-277. https://doi.org/10.5487/TR.2019.35.3.271
  15. Herman-Antosiewicz, A., Johnson, D. E. and Singh, S. V. (2006) Sulforaphane causes autophagy to inhibit release of cytochrome C and apoptosis in human prostate cancer cells. Cancer Res. 66, 5828-5835. https://doi.org/10.1158/0008-5472.CAN-06-0139
  16. Hwang-Bo, H., Jeong, J. W., Han, M. H., Park, C., Hong, S. H., Kim, G. Y., Moon, S. K., Cheong, J., Kim, W. J., Yoo, Y. H. and Choi, Y. H. (2017) Auranofin, an inhibitor of thioredoxin reductase, induces apoptosis in hepatocellular carcinoma Hep3B cells by generation of reactive oxygen species. Gen. Physiol. Biophys. 36, 117-128. https://doi.org/10.4149/gpb_2016043
  17. Hwang-Bo, H., Lee, W. S., Nagappan, A., Kim, H. J., Panchanathan, R., Park, C., Chang, S. H., Kim, N. D., Leem, S. H., Chang, Y. C., Kwon, T. K., Cheong, J. H., Kim, G. S., Jung, J. M., Shin, S. C., Hong, S. C. and Choi, Y. H. (2019) Morin enhances auranofin anticancer activity by up-regulation of DR4 and DR5 and modulation of Bcl-2 through reactive oxygen species generation in Hep3B human hepatocellular carcinoma cells. Phytother. Res. 33, 1384-1393. https://doi.org/10.1002/ptr.6329
  18. Isab, A. A. and Shaw, C. F., 3rd (1990) Synthesis of thionato(triethylphosphine) gold(I) complexes: analogues of "auranofin" an antiarthritic drug. J. Inorg. Biochem. 38, 95-100. https://doi.org/10.1016/0162-0134(90)84017-J
  19. Jia, J. J., Geng, W. S., Wang, Z. Q., Chen, L. and Zeng, X. S. (2019) The role of thioredoxin system in cancer: strategy for cancer therapy. Cancer Chemother. Pharmacol. 84, 453-470. https://doi.org/10.1007/s00280-019-03869-4
  20. Kerantzas, C. A. and Jacobs, W. R., Jr. (2017) Origins of combination therapy for tuberculosis: lessons for future antimicrobial development and application. mBio 8, e01586-16.
  21. Kulik, L. and El-Serag, H. B. (2019) Epidemiology and management of hepatocellular carcinoma. Gastroenterology 156, 477-491. https://doi.org/10.1053/j.gastro.2018.08.065
  22. Li, Y., Zhang, T., Korkaya, H., Liu, S., Lee, H. F., Newman, B., Yu, Y., Clouthier, S. G., Schwartz, S. J., Wicha, M. S. and Sun, D. (2010) Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells. Clin. Cancer Res. 16, 2580-2590. https://doi.org/10.1158/1078-0432.CCR-09-2937
  23. Likhitsup, A., Razumilava, N. and Parikh, N. D. (2019) Treatment for advanced hepatocellular carcinoma: current standard and the future. Clin. Liver Dis. (Hoboken) 13, 13-19. https://doi.org/10.1002/cld.782
  24. Lincoln, D. T., Ali, Emadi, E. M., Tonissen, K. F. and Clarke, F. M. (2003) The thioredoxin-thioredoxin reductase system: over-expression in human cancer. Anticancer Res. 23, 2425-2433.
  25. Llovet, J. M., Montal, R., Sia, D. and Finn, R. S. (2018) Molecular therapies and precision medicine for hepatocellular carcinoma. Nat. Rev. Clin. Oncol. 15, 599-616. https://doi.org/10.1038/s41571-018-0073-4
  26. Lu, J. and Holmgren, A. (2014) The thioredoxin antioxidant system. Free. Radic. Biol. Med. 66, 75-87. https://doi.org/10.1016/j.freeradbiomed.2013.07.036
  27. Madeira, J. M., Gibson, D. L., Kean, W. F. and Klegeris, A. (2012) The biological activity of auranofin: implications for novel treatment of diseases. Inflammopharmacology 20, 297-306. https://doi.org/10.1007/s10787-012-0149-1
  28. Marzano, C., Gandin, V., Folda, A., Scutari, G., Bindoli, A. and Rigobello, M. P. (2007) Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells. Free Radic. Biol. Med. 42, 872-881. https://doi.org/10.1016/j.freeradbiomed.2006.12.021
  29. McKelvey, E. M., Gottlieb, J. A., Wilson, H. E., Haut, A., Talley, R. W., Stephens, R., Lane, M., Gamble, J. F., Jones, S. E., Grozea, P. N., Gutterman, J., Coltman, C. and Moon, T. E. (1976) Hydroxyldaunomycin (Adriamycin) combination chemotherapy in malignant lymphoma. Cancer 38, 1484-1493. https://doi.org/10.1002/1097-0142(197610)38:4<1484::AID-CNCR2820380407>3.0.CO;2-I
  30. Mi, L., Wang, X., Govind, S., Hood, B. L., Veenstra, T. D., Conrads, T. P., Saha, D. T., Goldman, R. and Chung, F. L. (2007) The role of protein binding in induction of apoptosis by phenethyl isothiocyanate and sulforaphane in human non-small lung cancer cells. Cancer Res. 67, 6409-6416. https://doi.org/10.1158/0008-5472.CAN-07-0340
  31. Moon, D. O., Kang, S. H., Kim, K. C., Kim, M. O., Choi, Y. H. and Kim, G. Y. (2010) Sulforaphane decreases viability and telomerase activity in hepatocellular carcinoma Hep3B cells through the reactive oxygen species-dependent pathway. Cancer Lett. 295, 260-266. https://doi.org/10.1016/j.canlet.2010.03.009
  32. Niedzwiecki, A., Roomi, M. W., Kalinovsky, T. and Rath, M. (2016) Anticancer efficacy of polyphenols and their combinations. Nutrients 8, 552. https://doi.org/10.3390/nu8090552
  33. Omata, Y., Folan, M., Shaw, M., Messer, R. L., Lockwood, P. E., Hobbs, D., Bouillaguet, S., Sano, H., Lewis, J. B. and Wataha, J. C. (2006) Sublethal concentrations of diverse gold compounds inhibit mammalian cytosolic thioredoxin reductase (TrxR1). Toxicol. In Vitro 20, 882-890. https://doi.org/10.1016/j.tiv.2006.01.012
  34. Ouyang, Y., Peng, Y., Li, J., Holmgren, A. and Lu, J. (2018) Modulation of thiol-dependent redox system by metal ions via thioredoxin and glutaredoxin systems. Metallomics 10, 218-228. https://doi.org/10.1039/C7MT00327G
  35. Phan, M. A. T., Paterson, J., Bucknall, M. and Arcot, J. (2018) Interactions between phytochemicals from fruits and vegetables: effects on bioactivities and bioavailability. Crit. Rev. Food Sci. Nutr. 58, 1310-1329. https://doi.org/10.1080/10408398.2016.1254595
  36. Pritchard, J. R., Lauffenburger, D. A. and Hemann, M. T. (2012) Understanding resistance to combination chemotherapy. Drug Resist. Updat. 15, 249-257. https://doi.org/10.1016/j.drup.2012.10.003
  37. Ralph, S. J., Nozuhur, S., ALHulais, R. A., Rodriguez-Enriquez, S. and Moreno-Sanchez, R. (2019) Repurposing drugs as pro-oxidant redox modifiers to eliminate cancer stem cells and improve the treatment of advanced stage cancers. Med. Res. Rev. 39, 2397-2426. https://doi.org/10.1002/med.21589
  38. Ren, X., Zou, L., Lu, J. and Holmgren, A. (2018) Selenocysteine in mammalian thioredoxin reductase and application of ebselen as a therapeutic. Free Radic. Biol. Med. 127, 238-247. https://doi.org/10.1016/j.freeradbiomed.2018.05.081
  39. Robak, T., Blonski, J. Z. and Robak, P. (2016) Antibody therapy alone and in combination with targeted drugs in chronic lymphocytic leukemia. Semin. Oncol. 43, 280-290. https://doi.org/10.1053/j.seminoncol.2016.02.010
  40. Robbins, R. J., Keck, A. S., Banuelos, G. and Finley, J. W. (2005) Cultivation conditions and selenium fertilization alter the phenolic profile, glucosinolate, and sulforaphane content of broccoli. J. Med. Food 8, 204-214. https://doi.org/10.1089/jmf.2005.8.204
  41. Schirrmacher, V. (2019) From chemotherapy to biological therapy: a review of novel concepts to reduce the side effects of systemic cancer treatment (review). Int. J. Oncol. 54, 407-419.
  42. Trotti, A., Byhardt, R., Stetz, J., Gwede, C., Corn, B., Fu, K., Gunderson, L., McCormick, B., Morrisintegral, M., Rich, T., Shipley, W. and Curran, W. (2000) Common toxicity criteria: version 2.0. an improved reference for grading the acute effects of cancer treatment: impact on radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 47, 13-47. https://doi.org/10.1016/S0360-3016(99)00559-3
  43. Urig, S. and Becker, K. (2006) On the potential of thioredoxin reductase inhibitors for cancer therapy. Semin. Cancer Biol. 16, 452-465. https://doi.org/10.1016/j.semcancer.2006.09.004
  44. U.S. National Library of Medicine, ClinicalTrials.gov. Available from: https://clinicaltrials.gov/ct2/home/.
  45. Yu, J. S. and Cui, W. (2016) Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development 143, 3050-3060. https://doi.org/10.1242/dev.137075
  46. Zhang, N., Li, F., Gao, J., Zhang, S. and Wang, Q. (2020) Osteopontin accelerates the development and metastasis of bladder cancer via activating JAK1/STAT1 pathway. Genes Genomics 42, 467-475. https://doi.org/10.1007/s13258-019-00907-6
  47. Zhong, L., Arner, E. S. and Holmgren, A. (2000) Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence. Proc. Natl. Acad. Sci. U.S.A. 97, 5854-5859. https://doi.org/10.1073/pnas.100114897
  48. Zorova, L. D., Popkov, V. A., Plotnikov, E. Y., Silachev, D. N., Pevzner, I. B., Jankauskas, S. S., Babenko, V. A., Zorov, S. D., Balakireva, A. V., Juhaszova, M., Sollott, S. J. and Zorov, D. B. (2018) Mitochondrial membrane potential. Anal. Biochem. 552, 50-59. https://doi.org/10.1016/j.ab.2017.07.009

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