Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Induction of Apoptosis by Ethanol Extract of Cnidium officinale in Human Leukemia U937 Cells through Activation of AMPK

천궁 에탄올 추출물의 AMPK 활성화를 통한 U937 인체 혈구암세포의 apoptosis 유발

  • Jeong, Jin-Woo (Department of Biochemistry, Dongeui University College of Oriental Medicine) ;
  • Choi, Yung Hyun (Department of Biochemistry, Dongeui University College of Oriental Medicine) ;
  • Park, Cheol (Department of Molecular Biology, College of Natural Sciences and Human Ecology, Dongeui University)
  • 정진우 (동의대학교 한의과대학 생화학교실) ;
  • 최영현 (동의대학교 한의과대학 생화학교실) ;
  • 박철 (동의대학교 자연생활 과학대학 분자생물학과)
  • Received : 2015.08.07
  • Accepted : 2015.09.16
  • Published : 2015.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

Cnidium officinale, a traditional herb, has diverse beneficial pharmacological activities, such as anti-inflammatory, antioxidant, anticancer, and antiangiogenesis effects. However, the cellular and molecular mechanisms of apoptosis by C. officinale are poorly defined. The present study investigated the proapoptotic effects of water, ethanol, and methanol extract of C. officinale (WECO, EECO, and MECO, respectively) in human leukemia U937 cells. The antiproliferative activity of EECO was higher than that of WECO and MECO. The antiproliferative effect of EECO treatment in U937 cells was associated with the induction of apoptotic cell death, including increased populations of annexin-V positive cells, the formation of apoptotic bodies, DNA fragmentation, and increased numbers of cells with a loss of mitochondrial membrane potential (MMP, Δψm). EECO-induced apoptotic cell death was associated with upregulation of death receptor 4 (DR4) and down-regulation of cellular inhibitor of apoptosis protein-1 (cIAP-1), Bcl-2, and total Bid. The EECO treatment also induced the proteolytic activation of caspases (-3, -8, and -9), and degradation of caspase-3 substrate proteins, such as poly(ADP-ribose) polymerase (PARP), β-catenin, and phospholipase C-γ1 (PLCγ1). In addition, the EECO treatment effectively activated the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. However, compound C, a specific inhibitor of AMPK, significantly reduced EECO-induced apoptosis. These results indicate that AMPK is a key regulator of apoptosis in response to EECO in human leukemia U937 cells.

천궁(C. officinale)은 예로부터 민간처방 약재로 사용되었으며, 항염증, 항산화, 항암 및 신생혈관억제 등의 효능을 가지는 것으로 알려져 있다. 하지만 혈구암세포에서 apoptosis 유발과 관련된 분자생물학적 기전에 대해서는 명확히 밝혀져 있지 않다. 본 연구에서는 인체 혈구암세포인 U937 세포에서 천궁의 열수, 에탄올 및 메탄올 추출물(WECO, EECO 및 MECO)이 유발하는 항암효과 및 항암기전을 조사하였다. 먼저 WECO, EECO 및 MECO가 유발하는 증식억제 정도를 조사한 결과 EECO가 가장 뛰어난 효능을 가진다는 것을 알 수 있었으며, 이러한 현상이 apoptosis 유발에 의한 것임을 annexin-V 염색, apoptotic body 형성, DNA 단편화 및 MMP 소실 등을 통하여 확인하였다. EECO 처리에 의한 apoptosis 유발에는 DR4의 발현 증가와 함께 cIAP-1, Bcl-2 및 total Bid의 발현감소가 관여하였으며, caspases-3, -8 및 -9의 활성화와 함께 caspases-3의 기질 단백질인 PARP, β-catenin 및 PLC γ1의 단편화도 관찰되었다. 또한 EECO는 AMPK signaling pathway를 활성화시키는 것으로 나타났으며, AMPK 억제제인 compound C를 이용하여 AMPK의 활성을 억제하였을 경우 EECO에 의하여 유발되었던 apoptosis가 현저하게 감소되는 것으로 나타났다. 이상의 결과를 살펴볼 때 인체 혈구암세포인 U937 세포에서 EECO에 의하여 유발되는 apoptosis는 AMPK가 중요한 조절자로서 작용하는 것으로 생각된다.

Keywords

References

  1. Bae, S. S., Perry, D. K., Oh, Y. S., Choi, J. H., Galadari, S. H., Ghayur, T., Ryu, S. H., Hannun, Y. A. and Suh, P. G. 2000. Proteolytic cleavage of phospholipase C-gamma1 during apoptosis in Molt-4 cells. FASEB J. 14, 1083-1092.
  2. Brunelle, J. K. and Letai, A. 2009. Control of mitochondrial apoptosis by the Bcl-2 family. J. Cell Sci. 122, 437-441. https://doi.org/10.1242/jcs.031682
  3. Chen, S., Xiao, X., Feng, X., Li, W., Zhou, N., Zheng, L., Sun, Y., Zhang, Z. and Zhu, W. 2012. Resveratrol induces Sirt1-dependent apoptosis in 3T3-L1 preadipocytes by activating AMPK and suppressing AKT activity and survivin expression. J. Nutr. Biochem. 23, 1100-1112. https://doi.org/10.1016/j.jnutbio.2011.06.003
  4. de Almagro, M. C. and Vucic, D. 2012. The inhibitor of apoptosis (IAP) proteins are critical regulators of signaling pathways and targets for anti-cancer therapy. Exp. Oncol. 34, 200-211.
  5. de la Cruz, J., Kim, D. H. and Hwang, S. G. 2014. Anti cancer effects of Cnidium officinale Makino extract mediated through apoptosis and cell cycle arrest in the HT-29 human colorectal cancer cell line. Asian Pac. J. Cancer Prev. 15, 5117-5121. https://doi.org/10.7314/APJCP.2014.15.13.5117
  6. Fiandalo, M. V. and Kyprianou, N. 2012. Caspase control: protagonists of cancer cell apoptosis. Exp. Oncol. 34, 165-175.
  7. Fogarty, S. and Hardie, D. G. 2010. Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer. Biochim. Biophys. Acta. 1804, 581-591. https://doi.org/10.1016/j.bbapap.2009.09.012
  8. Fukuda, K. 1999. Apoptosis-associated cleavage of beta-catenin in human colon cancer and rat hepatoma cells. Int. J. Biochem. Cell Biol. 31, 519-529. https://doi.org/10.1016/S1357-2725(98)00119-8
  9. Fulda, S. and Debatin, K. M. 2006. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798-4811. https://doi.org/10.1038/sj.onc.1209608
  10. Han, S. I., Kim, Y. S. and Kim, T. H. 2008. Role of apoptotic and necrotic cell death under physiologic conditions. BMB Rep. 41, 1-10. https://doi.org/10.5483/BMBRep.2008.41.1.001
  11. Hardie, D. G., Hawley, S. A. and Scott, J. W. 2006. AMP-activated protein kinase--development of the energy sensor concept. J. Physiol. 574, 7-15. https://doi.org/10.1113/jphysiol.2006.108944
  12. Jeong, J. B., Ju, S. Y., Park, J. H., Lee, J. R., Yun, K. W., Kwon, S. T., Lim, J. H., Chung, G. Y. and Jeong, H. J. 2009. Antioxidant activity in essential oils of Cnidium officinale makino and Ligusticum chuanxiong Hort and their inhibitory effects on DNA damage and apoptosis induced by ultraviolet B in mammalian cell. Cancer Epidemiol. 33, 41-46. https://doi.org/10.1016/j.canep.2009.04.010
  13. Jin, Z. and El-Deiry, W. S. 2005. Overview of cell death signaling pathways. Cancer Biol. Ther. 4, 139-163.
  14. Kwak, D. H., Kim, J. K., Kim, J. Y., Jeong, H. Y., Keum, K. S., Han, S. H., Rho, Y. I., Woo, W. H., Jung, K. Y., Choi, B. K. and Choo, Y. K. 2002. Anti-angiogenic activities of Cnidium officinale Makino and Tabanus bovinus. J. Ethnopharmacol. 81, 373-379. https://doi.org/10.1016/S0378-8741(02)00122-8
  15. Lee, D. H., Lee, T. H., Jung, C. H. and Kim, Y. H. 2012. Wogonin induces apoptosis by activating the AMPK and p53 signaling pathways in human glioblastoma cells. Cell. Signal. 24, 2216-2225. https://doi.org/10.1016/j.cellsig.2012.07.019
  16. Lee, S. H., Lee, J. H., Oh, E. Y., Kim, G. Y., Choi, B. T., Kim, C. and Choi, Y. H. 2013. Ethanol extract of Cnidium officinale exhibits anti-inflammatory effects in BV2 microglial cells by suppressing NF-κB nuclear translocation and the activation of the PI3K/Akt signaling pathway. Int. J. Mol. Med. 32, 876-882.
  17. Li, H., Zhu, H., Xu, C. J. and Yuan, J. 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491-501 https://doi.org/10.1016/S0092-8674(00)81590-1
  18. Lopez-Beltran, A., Maclennan, G. T., de la Haba-Rodriguez, J., Montironi, R. and Cheng, L. 2007. Research advances in apoptosis-mediated cancer therapy: a review. Anal. Quant. Cytol. Histol. 29, 71-78.
  19. Martinou, J. C. and Youle, R. J. 2011. Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev. Cell 21, 92-101. https://doi.org/10.1016/j.devcel.2011.06.017
  20. Momcilovic, M., Hong, S. P. and Carlson, M. 2006. Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro. J. Biol. Chem. 281, 25336-25343. https://doi.org/10.1074/jbc.M604399200
  21. Okada, H. and Mak, T. W. 2004. Pathways of apoptotic and non-apoptotic death in tumour cells. Nat. Rev. Cancer 4, 592-603. https://doi.org/10.1038/nrc1412
  22. Ozaki, Y., Sekita, S. and Harada, M. 1989. Centrally acting muscle relaxant effect of phthalides (ligustilide, cnidilide and senkyunolide) obtained from Cnidium officinale Makino. Yakugaku Zasshi 109, 402-406.
  23. Peter, M. E. and Krammer, P. H. 2003. The CD95 (APO-1/Fas) DISC and beyond. Cell Death Differ. 10, 26-35. https://doi.org/10.1038/sj.cdd.4401186
  24. Shen, Q. W., Zhu, M. J., Tong, J., Ren, J. and Du, M. 2007. Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by alpha-lipoic acid in C2C12 myotubes. Am. J. Physiol. Cell Physiol. 293, C1395-1403. https://doi.org/10.1152/ajpcell.00115.2007
  25. Tiainen, M., Vaahtomeri, K., Ylikorkala, A. and Mäkelä, T. P. 2002. Growth arrest by the LKB1 tumor suppressor: induction of p21(WAF1/CIP1). Hum. Mol. Genet. 11, 1497-1504. https://doi.org/10.1093/hmg/11.13.1497
  26. Tomoda, M., Ohara, N., Gonda, R., Shimizu, N., Takada, K., Satoh, Y. and Shirai, S. 1992. An acidic polysaccharide having immunological activities from the rhizome of Cnidium officinale. Chem. Pharm. Bull. (Tokyo) 40, 3025-3029. https://doi.org/10.1248/cpb.40.3025
  27. Vilches Troya, J. 2005. Understanding cell death: a challenge for biomedicine. An. R. Acad. Nac. Med. (Madr) 122, 631-656.
  28. Wu, S. J., Huang, G. Y. and Ng, L. T. 2013. γ-Tocotrienol induced cell cycle arrest and apoptosis via activating the Bax-mediated mitochondrial and AMPK signaling pathways in 3T3-L1 adipocytes. Food Chem. Toxicol. 59, 501-513. https://doi.org/10.1016/j.fct.2013.06.011
  29. Yi, B., Liu, D., He, M., Li, Q., Liu, T. and Shao, J. 2013. Role of the ROS/AMPK signaling pathway in tetramethylpyrazine-induced apoptosis in gastric cancer cells. Oncol. Lett. 6, 583-589.
  30. Yinjun, L., Jie, J., Weilai, X. and Xiangming, T. 2004. Homoharringtonine mediates myeloid cell apoptosis via upregulation of pro-apoptotic bax and inducing caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). Am. J. Hematol. 76, 199-204. https://doi.org/10.1002/ajh.20100
  31. Yoshida, A., Pommier, Y. and Ueda, T. 2006. Endonuclease activation and chromosomal DNA fragmentation during apoptosis in leukemia cells. Int. J. Hematol. 84, 31-37. https://doi.org/10.1007/BF03342699
  32. Zeqiraj, E., Filippi, B. M., Deak, M., Alessi, D. R. and van Aalten, D. M. 2009. Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation. Science 326, 1707-1711. https://doi.org/10.1126/science.1178377

Cited by

  1. Effect of explant type and plant growth regulators on callus induction, growth and secondary metabolites production in Cnidium officinale Makino pp.1573-4978, 2018, https://doi.org/10.1007/s11033-018-4340-3