Journal of Ginseng Research

The Korean Society of Ginseng (고려인삼학회)
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Induction of Forkhead Class box O3a and apoptosis by a standardized ginsenoside formulation, KG-135, is potentiated by autophagy blockade in A549 human lung cancer cells

  • Yao, Chih-Jung (Cancer Center, Wan Fang Hospital, Taipei Medical University) ;
  • Chow, Jyh-Ming (Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University) ;
  • Chuang, Shuang-En (National Institute of Cancer Research, National Health Research Institutes) ;
  • Chang, Chia-Lun (Division of Hematology and Medical Oncology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University) ;
  • Yan, Ming-De (Cancer Center, Wan Fang Hospital, Taipei Medical University) ;
  • Lee, Hsin-Lun (Translational Medicine, Taipei Medical University-Academia Sinica) ;
  • Lai, I-Chun (Department of Oncology, Taipei Veterans General Hospital) ;
  • Lin, Pei-Chun (Cancer Center, Wan Fang Hospital, Taipei Medical University) ;
  • Lai, Gi-Ming (Cancer Center, Wan Fang Hospital, Taipei Medical University)
  • Received : 2015.11.13
  • Accepted : 2016.04.06
  • Published : 2017.07.15
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Abstract

Background: KG-135, a standardized formulation enriched with Rk1, Rg3, and Rg5 ginsenosides, has been shown to inhibit various types of cancer cells; however, the underlying mechanisms are not fully understood. In this study, we explored its effects in A549 human lung cancer cells to investigate the induction of Forkhead Class box O3a (FOXO3a) and autophagy. Methods: Cell viability was determined by sulforhodamine B staining. Apoptosis and cell cycle distribution were analyzed using flow cytometry. The changes of protein levels were determined using Western blot analysis. Autophagy induction was monitored by the formation of acidic vesicular organelles stained with acridine orange. Results: KG-135 effectively arrested the cells in G1 phase with limited apoptosis. Accordingly, a decrease of cyclin-dependent kinase-4, cyclin-dependent kinase-6, cyclin D1, and phospho-retinoblastoma protein, and an increase of p27 and p18 proteins were observed. Intriguingly, KG-135 increased the tumor suppressor FOXO3a and induced the accumulation of autophagy hallmark LC3-II and acidic vesicular organelles without an increase of the upstream marker Beclin-1. Unconventionally, the autophagy adaptor protein p62 (sequestosome 1) was increased rather than decreased. Blockade of autophagy by hydroxychloroquine dramatically potentiated KG-135-induced FOXO3a and its downstream (FasL) ligand accompanied by the cleavage of caspase-8. Meanwhile, the decrease of Bcl-2 and survivin, as well as the cleavage of caspase-9, were also drastically enhanced, resulting in massive apoptosis. Conclusion: Besides arresting the cells in G1 phase, KG-135 increased FOXO3a and induced an unconventional autophagy in A549 cells. Both the KG-135-activated extrinsic FOXO3a/FasL/caspase-8 and intrinsic caspase-9 apoptotic pathways were potentiated by blockade of autophagy. Combination of KG-135 and autophagy inhibitor may be a novel strategy as an integrative treatment for cancers.

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References

  1. Xiang YZ, Shang HC, Gao XM, Zhang BL. A comparison of the ancient use of ginseng in traditional Chinese medicine with modern pharmacological experiments and clinical trials. Phytother Res 2008;22:851-8. https://doi.org/10.1002/ptr.2384
  2. Choi KT. Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng Meyer. Acta Pharmacol Sin 2008;29:1109-18. https://doi.org/10.1111/j.1745-7254.2008.00869.x
  3. Wong AS, Che CM, Leung KW. Recent advances in ginseng as cancer therapeutics: a functional and mechanistic overview. Nat Prod Rep 2015;32:256-72. https://doi.org/10.1039/C4NP00080C
  4. Choi HJ, Kim EJ, Shin YW, Park JH, Kim DH, Kim NJ. Protective effect of heatprocessed ginseng (sun ginseng) in the adenine-induced renal failure rats. J Ginseng Res 2012;36:270-6. https://doi.org/10.5142/jgr.2012.36.3.270
  5. Lee WH, Choi JS, Kim HY, Park JH, Lee SK, Surh YJ. Heat-processed neoginseng, KG-135, down-regulates G1 cyclin-dependent kinase through the proteasome-mediated pathway in HeLa cells. Oncol Rep 2009;21:467-74.
  6. Wang CZ, Aung HH, Ni M, Wu JA, Tong R, Wicks S, He TC, Yuan CS. Red American ginseng: ginsenoside constituents and antiproliferative activities of heat-processed Panax quinquefolius roots. Planta Med 2007;73:669-74. https://doi.org/10.1055/s-2007-981524
  7. Yun TK, Lee YS, Lee YH, Kim SI, Yun HY. Anticarcinogenic effect of Panax ginseng C.A. Meyer and identification of active compounds. J Korean Med Sci 2001;16(Suppl):S6-18. https://doi.org/10.3346/jkms.2001.16.S.S6
  8. Yoo JH, Kwon HC, Kim YJ, Park JH, Yang HO. KG-135, enriched with selected ginsenosides, inhibits the proliferation of human prostate cancer cells in culture and inhibits xenograft growth in athymic mice. Cancer Lett 2010;289:99-110. https://doi.org/10.1016/j.canlet.2009.08.008
  9. Park JY, Choi P, Kim HK, Kang KS, Ham J. Increase in apoptotic effect of Panax ginseng by microwave processing in human prostate cancer cells: in vitro and in vivo studies. J Ginseng Res 2016;40:62-7. https://doi.org/10.1016/j.jgr.2015.04.007
  10. Park SA, Kim EH, Na HK, Surh YJ. KG-135 inhibits COX-2 expression by blocking the activation of JNK and AP-1 in phorbol ester-stimulated human breast epithelial cells. Ann N Y Acad Sci 2007;1095:545-53. https://doi.org/10.1196/annals.1397.059
  11. Lee WH, Choi JS, Kim HY, Park JH, Park BD, Cho SJ, Lee SK, Surh YJ. Potentiation of etoposide-induced apoptosis in HeLa cells by co-treatment with KG-135, a quality-controlled standardized ginsenoside formulation. Cancer Lett 2010;294:74-81. https://doi.org/10.1016/j.canlet.2010.01.024
  12. Mizushima N. Autophagy: process and function. Genes Dev 2007;21:2861-73. https://doi.org/10.1101/gad.1599207
  13. Eskelinen EL, Saftig P. Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta 2009;1793:664-73. https://doi.org/10.1016/j.bbamcr.2008.07.014
  14. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004;36:2491-502. https://doi.org/10.1016/j.biocel.2004.02.005
  15. de Bruin EC, Medema JP. Apoptosis and nonapoptotic deaths in cancer development and treatment response. Cancer Treat Rev 2008;34:737-49. https://doi.org/10.1016/j.ctrv.2008.07.001
  16. Mizushima N. The pleiotropic role of autophagy: from protein metabolism to bactericide. Cell Death Differ 2005;12(Suppl. 2):1535-41. https://doi.org/10.1038/sj.cdd.4401728
  17. Kaminskyy VO, Piskunova T, Zborovskaya IB, Tchevkina EM, Zhivotovsky B. Suppression of basal autophagy reduces lung cancer cell proliferation and enhances caspase-dependent and -independent apoptosis by stimulating ROS formation. Autophagy 2012;8:1032-44. https://doi.org/10.4161/auto.20123
  18. Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, Han W, Lou F, Yang J, Zhang Q, et al. Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis 2013;4:1-12.
  19. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005;5:726-34. https://doi.org/10.1038/nrc1692
  20. Cheong JH, Kim H, Hong MJ, Yang MH, Kim JW, Yoo H, Yang H, Park JH, Sung SH, Kim HP, et al. Stereoisomer-specific anticancer activities of ginsenoside Rg3 and Rh2 in HepG2 cells: disparity in cytotoxicity and autophagy-inducing effects due to 20(S)-epimers. Biol Pharm Bull 2015;38:102-8.
  21. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893-917. https://doi.org/10.1002/ijc.25516
  22. Bartucci M, Svensson S, Romania P, Dattilo R, Patrizii M, Signore M, Navarra S, Lotti F, Biffoni M, Pilozzi E, et al. Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy. Cell Death Differ 2012;19:768-78. https://doi.org/10.1038/cdd.2011.170
  23. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277-300. https://doi.org/10.3322/caac.20073
  24. Collins LG, Haines C, Perkel R, Enck RE. Lung cancer: diagnosis and management. Am Fam Physician 2007;75:56-63.
  25. Wang L, Li X, Song YM, Wang B, Zhang FR, Yang R, Wang HQ, Zhang GJ. Ginsenoside Rg3 sensitizes human nonsmall cell lung cancer cells to gammaradiation by targeting the nuclear factor-kappaB pathway. Mol Med Rep 2015;12:609-14. https://doi.org/10.3892/mmr.2015.3397
  26. Kim YJ, Choi WI, Jeon BN, Choi KC, Kim K, Kim TJ, Ham J, Jang HJ, Kang KS, Ko H. Stereospecific effects of ginsenoside 20-Rg3 inhibits TGF-beta1-induced epithelial-mesenchymal transition and suppresses lung cancer migration, invasion and anoikis resistance. Toxicology 2014;322:23-33. https://doi.org/10.1016/j.tox.2014.04.002
  27. Bi X, Xia X, Mou T, Jiang B, Fan D, Wang P, Liu Y, Hou Y, Zhao Y. Antitumor activity of three ginsenoside derivatives in lung cancer is associated with Wnt/beta-catenin signaling inhibition. Eur J Pharmacol 2014;742:145-52. https://doi.org/10.1016/j.ejphar.2014.08.032
  28. Zhang LH, Jia YL, Lin XX, Zhang HQ, Dong XW, Zhao JM, Shen J, Shen HJ, Li FF, Yan XF, et al. AD-1, a novel ginsenoside derivative, shows anti-lung cancer activity via activation of p38 MAPK pathway and generation of reactive oxygen species. Biochim Biophys Acta 2013;1830:4148-59. https://doi.org/10.1016/j.bbagen.2013.04.008
  29. An IS, An S, Kwon KJ, Kim YJ, Bae S. Ginsenoside Rh2 mediates changes in the microRNA expression profile of human non-small cell lung cancer A549 cells. Oncol Rep 2013;29:523-8. https://doi.org/10.3892/or.2012.2136
  30. Cheng CC, Yang SM, Huang CY, Chen JC, Chang WM, Hsu SL. Molecular mechanisms of ginsenoside Rh2-mediated G1 growth arrest and apoptosis in human lung adenocarcinoma A549 cells. Cancer Chemother Pharmacol 2005;55:531-40. https://doi.org/10.1007/s00280-004-0919-6
  31. Ruvolo PP. The Herculean task of killing cancer cells: suppression of FOXO3A in acute leukemia involves a hydra of multiple survival kinases. Cell Cycle 2012;11:2589. https://doi.org/10.4161/cc.21233
  32. Garcia-Navas R, Munder M, Mollinedo F. Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic reticulum stress in human T lymphocytes. Autophagy 2012;8:1557-76. https://doi.org/10.4161/auto.21315
  33. Traganos F, Darzynkiewicz Z. Lysosomal proton pump activity: supravital cell staining with acridine orange differentiates leukocyte subpopulations. Methods Cell Biol 1994;41:185-94.
  34. Zhao Y, Fei M, Wang Y, Lu M, Cheng C, Shen A. Expression of Foxo3a in non-Hodgkin's lymphomas is correlated with cell cycle inhibitor p27. Eur J Haematol 2008;81:83-93. https://doi.org/10.1111/j.1600-0609.2008.01077.x
  35. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012;8:445-544. https://doi.org/10.4161/auto.19496
  36. Feng Y, Ke C, Tang Q, Dong H, Zheng X, Lin W, Ke J, Huang J, Yeung SC, Zhang H. Metformin promotes autophagy and apoptosis in esophageal squamous cell carcinoma by downregulating Stat3 signaling. Cell Death Dis 2014;5:e1088. https://doi.org/10.1038/cddis.2014.59
  37. Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy 2007;3:542-5. https://doi.org/10.4161/auto.4600
  38. Sasaki K, Tsuno NH, Sunami E, Tsurita G, Kawai K, Okaji Y, Nishikawa T, Shuno Y, Hongo K, Hiyoshi M, et al. Chloroquine potentiates the anti-cancer effect of 5-fluorouracil on colon cancer cells. BMC Cancer 2010;10:370. https://doi.org/10.1186/1471-2407-10-370
  39. Gunja N, Roberts D, McCoubrie D, Lamberth P, Jan A, Simes DC, Hackett P, Buckley NA. Survival after massive hydroxychloroquine overdose. Anaesth Intensive Care 2009;37:130-3.
  40. Codogno P, Mehrpour M, Proikas-Cezanne T. Canonical and noncanonical autophagy: variations on a common theme of self-eating? Nat Rev Mol Cell Biol 2012;13:7-12.
  41. Puissant A, Robert G, Fenouille N, Luciano F, Cassuto JP, Raynaud S, Auberger P. Resveratrol promotes autophagic cell death in chronic myelogenous leukemia cells via JNK-mediated p62/SQSTM1 expression and AMPK activation. Cancer Res 2010;70:1042-52. https://doi.org/10.1158/0008-5472.CAN-09-3537
  42. Puissant A, Auberger P. AMPK- and p62/SQSTM1-dependent autophagy mediate resveratrol-induced cell death in chronic myelogenous leukemia. Autophagy 2010;6:655-7. https://doi.org/10.4161/auto.6.5.12126
  43. Kim DG, Jung KH, Lee DG, Yoon JH, Choi KS, Kwon SW, Shen HM, Morgan MJ, Hong SS, Kim YS. 20(S)-Ginsenoside Rg3 is a novel inhibitor of autophagy and sensitizes hepatocellular carcinoma todoxorubicin.Oncotarget2014;5:4438-51. https://doi.org/10.18632/oncotarget.2034

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