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Antiproliferative effect of Citrus junos extracts on A549 human non-small-cell lung cancer cells

  • Geum-Bi, Ryu (Department of Food and Nutrition, Chonnam National University) ;
  • Young-Ran, Heo (Division of Food and Nutrition, Chonnam National University)
  • Received : 2023.01.16
  • Accepted : 2023.02.03
  • Published : 2023.02.28

Abstract

Purpose: This study investigates the alterations in A549 human non-small-cell lung cancer (NSCLC) cells exposed to Citrus junos extract (CJE). We further examine the antiproliferative and apoptotic effects of CJE on NSCLC cells. Methods: Inhibition of proliferation was examined by applying the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) colorimetric assay on CJE-treated A549 NSCLC cells. The lactate dehydrogenase (LDH) assay was performed to measure the degree of toxicity of CJE on NSCLC cells. The effect on migratory proliferation was confirmed using the scratch wound healing assay. The antiproliferative effect of the CJE on human lung cancer cells was verified through morphological observation, fluorescence microscopy, and caspase-3 colorimetry. Results: Exposure of NSCLC cells to CJE resulted in a dose- and time-dependent decrease in cell activity and increased toxicity to the cells. In addition, microscopic observation revealed a reduced ability of the cancer cells to migrate and proliferate after exposure to the CJE, with simultaneous morphological apoptotic changes. Fluorescence staining and microscopic examination revealed that this death was a process of self-programmed cell death of NSCLC cells. Compared to unexposed NSCLC cells, the expression of caspase-3 was significantly increased in cells exposed to CJE. Conclusion: Exposure of A549 human NSCLC cells to CJE inhibits the proliferation, increases the cytotoxicity, and decreases the ability of cells to migrate and grow. Moreover, the expression of caspase-3 increases after CJE treatment, suggesting that the apoptosis of NSCLC cells is induced by a chain reaction initiated by caspase-3. These results indicate that Citrus junos is a potential therapeutic agent for human non-small-cell lung cancer.

Keywords

References

  1. Ferguson L, Grafton-Cardwell EE. Citrus production manual. Los Angeles (CA): UCANR Publications; 2014. 
  2. Gmitter FG, Hu X. The possible role of Yunnan, China, in the origin of contemporary citrus species (Rutaceae). Econ Bot 1990; 44(2): 267-277. https://doi.org/10.1007/BF02860491
  3. Hirota R, Roger NN, Nakamura H, Song HS, Sawamura M, Suganuma N. Anti-inflammatory effects of limonene from yuzu (Citrus junos Tanaka) essential oil on eosinophils. J Food Sci 2010; 75(3): H87-H92. https://doi.org/10.1111/j.1750-3841.2010.01541.x
  4. Yoo KM, Hwang IK. In vitro effect of Yuza (Citrus junos Sieb ex TANAKA) extracts on proliferation of human prostate cancer cells and antioxidant activity. Korean J Food Sci Technol 2004; 36(2): 339-344. 
  5. Kim K, Cho H, Jung H, Lee HJ, Hwang KT. Anti-proliferative effect of methanolic extracts from Citrus junos seeds and seed oils on HT-29 human colon cancer cells and identification of their major bioactive compounds. Korean J Food Sci Technol 2017; 49(3): 242-251. https://doi.org/10.9721/KJFST.2017.49.3.242
  6. Kim SH, Shin EJ, Hur HJ, Park JH, Sung MJ, Kwon DY, et al. Citrus junos Tanaka peel extract attenuates experimental colitis and inhibits tumor growth in a mouse xenograft model. J Funct Foods 2014; 8(1): 301-308. https://doi.org/10.1016/j.jff.2014.03.024
  7. Park JH. Anti-diabetic and anticancer effects of peel of Citrus junos and Poncirus trifoliata [dissertation]. Busan: Dong-Eui University; 2008. 
  8. Shin EJ, Park JH, Sung MJ, Chung MY, Hwang JT. Citrus junos Tanaka peel ameliorates hepatic lipid accumulation in HepG2 cells and in mice fed a high-cholesterol diet. BMC Complement Altern Med 2016; 16(1): 499-507. https://doi.org/10.1186/s12906-016-1460-y
  9. Yoo KM, Lee KW, Park JB, Lee HJ, Hwang IK. Variation in major antioxidants and total antioxidant activity of Yuzu (Citrus junos Sieb ex Tanaka) during maturation and between cultivars. J Agric Food Chem 2004; 52(19): 5907-5913. https://doi.org/10.1021/jf0498158
  10. Shin Y. Correlation between antioxidant concentrations and activities of Yuja (Citrus junos Sieb ex Tanaka) and other citrus fruit. Food Sci Biotechnol 2012; 21(5): 1477-1482. https://doi.org/10.1007/s10068-012-0195-x
  11. Kim SH, Hur HJ, Yang HJ, Kim HJ, Kim MJ, Park JH, et al. Citrus junos Tanaka peel extract exerts antidiabetic effects via AMPK and PPAR-γ both in vitro and in vivo in mice fed a high-fat diet. Evid Based Complement Alternat Med 2013; 2013: 921012.
  12. Hwang JT, Yang HJ, Ha KC, So BO, Choi EK, Chae SW. A randomized, double-blind, placebo-controlled clinical trial to investigate the anti-diabetic effect of Citrus junos Tanaka peel. J Funct Foods 2015; 18(1): 532-537. https://doi.org/10.1016/j.jff.2015.08.019
  13. Zang L, Shimada Y, Kawajiri J, Tanaka T, Nishimura N. Effect of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet-induced obesity in a zebrafish model. J Funct Foods 2014; 10(1): 499-510. https://doi.org/10.1016/j.jff.2014.08.002
  14. Kaur J, Kaur G. An insight into the role of citrus bioactives in modulation of colon cancer. J Funct Foods 2015; 13(1): 239-261. https://doi.org/10.1016/j.jff.2014.12.043
  15. Heilmann J. New medical applications of plant secondary metabolites. Annu Plant Rev Online 2009; 39(2): 348-380.  https://doi.org/10.1002/9781119312994.apr0421
  16. Birsu Cincin Z, Unlu M, Kiran B, Sinem Bireller E, Baran Y, Cakmakoglu B. Anti-proliferative, apoptotic and signal transduction effects of hesperidin in non-small cell lung cancer cells. Cell Oncol (Dordr) 2015; 38(3): 195-204. https://doi.org/10.1007/s13402-015-0222-z
  17. Lien LM, Wang MJ, Chen RJ, Chiu HC, Wu JL, Shen MY, et al. Nobiletin, a polymethoxylated flavone, inhibits glioma cell growth and migration via arresting cell cycle and suppressing MAPK and Akt pathways. Phytother Res 2016; 30(2): 214-221. https://doi.org/10.1002/ptr.5517
  18. Jin CY, Park C, Hwang HJ, Kim GY, Choi BT, Kim WJ, et al. Naringenin up-regulates the expression of death receptor 5 and enhances TRAIL-induced apoptosis in human lung cancer A549 cells. Mol Nutr Food Res 2011; 55(2): 300-309. https://doi.org/10.1002/mnfr.201000024
  19. Xingyu Z, Peijie M, Dan P, Youg W, Daojun W, Xinzheng C, et al. Quercetin suppresses lung cancer growth by targeting Aurora B kinase. Cancer Med 2016; 5(11): 3156-3165. https://doi.org/10.1002/cam4.891
  20. Hung JY, Hsu YL, Ko YC, Tsai YM, Yang CJ, Huang MS, et al. Didymin, a dietary flavonoid glycoside from citrus fruits, induces Fas-mediated apoptotic pathway in human non-small-cell lung cancer cells in vitro and in vivo. Lung Cancer 2010; 68(3): 366-374. https://doi.org/10.1016/j.lungcan.2009.08.013
  21. Nguyen TTT, Tran E, Ong CK, Lee SK, Do PT, Huynh TT, et al. Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK-MAPK. J Cell Physiol 2003; 197(1): 110-121. https://doi.org/10.1002/jcp.10340
  22. Travis WD. Classification of lung cancer. Semin Roentgenol 2011; 46(3): 178-186. https://doi.org/10.1053/j.ro.2011.02.003
  23. Youlden DR, Cramb SM, Baade PD. The International Epidemiology of Lung Cancer: geographical distribution and secular trends. J Thorac Oncol 2008; 3(8): 819-831. https://doi.org/10.1097/jto.0b013e31818020eb
  24. Peters S, Adjei AA, Gridelli C, Reck M, Kerr K, Felip E, et al. Metastatic non-small-cell lung cancer (NSCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2012; 23 Suppl 7: vii56-vii64. https://doi.org/10.1093/annonc/mds226
  25. Gajra A, Newman N, Gamble GP, Abraham NZ, Kohman LJ, Graziano SL. Impact of tumor size on survival in stage IA non-small cell lung cancer: a case for subdividing stage IA disease. Lung Cancer 2003; 42(1): 51-57. https://doi.org/10.1016/S0169-5002(03)00285-X
  26. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267(5203): 1456-1462. https://doi.org/10.1126/science.7878464
  27. Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer 2002; 2(4): 277-288. https://doi.org/10.1038/nrc776
  28. Fesik SW. Promoting apoptosis as a strategy for cancer drug discovery. Nat Rev Cancer 2005; 5(11): 876-885. https://doi.org/10.1038/nrc1736
  29. Fan TJ, Han LH, Cong RS, Liang J. Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai) 2005; 37(11): 719-727. https://doi.org/10.1111/j.1745-7270.2005.00108.x
  30. Kitanaka C, Kuchino Y. Caspase-independent programmed cell death with necrotic morphology. Cell Death Differ 1999; 6(6): 508-515. https://doi.org/10.1038/sj/cdd/4400526
  31. Ernst O, Zor T. Linearization of the bradford protein assay. J Vis Exp 2010; 38(38): 1-6.
  32. Lenardo MJ, McPhee CK, Yu L. Autophagic cell death. Methods Enzymol 2009; 453(1): 17-31. https://doi.org/10.1016/S0076-6879(08)04002-0
  33. Patil JR, Jayaprakasha GK, Murthy KN, Tichy SE, Chetti MB, Patil BS. Apoptosis-mediated proliferation inhibition of human colon cancer cells by volatile principles of Citrus aurantifolia. Food Chem 2009; 114(4): 1351-1358. https://doi.org/10.1016/j.foodchem.2008.11.033