Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Anti-invasion Effects of Calystegia soldanella Solvent Extracts and Partitioned Fractions on PMA-stimulated Fibrosarcoma Cells

갯메꽃 추출물과 유기용매 분획물의 암전이 억제 효과

  • Son, Jaemin (Ocean Science and Technology School, Korea Maritime and Ocean University) ;
  • Kim, Junse (Ocean Science and Technology School, Korea Maritime and Ocean University) ;
  • Kim, Hojun (Division of Marine Bioscience, Korea Maritime and Ocean University) ;
  • Seo, Youngwan (Ocean Science and Technology School, Korea Maritime and Ocean University)
  • 손재민 (한국해양대학교 해양과학기술전문대학원 해양과학기술융합학과) ;
  • 김준세 (한국해양대학교 해양과학기술전문대학원 해양과학기술융합학과) ;
  • 김호준 (한국해양대학교 해양과학기술대학 해양환경.생명과학부) ;
  • 서영완 (한국해양대학교 해양과학기술전문대학원 해양과학기술융합학과)
  • Received : 2018.10.25
  • Accepted : 2018.11.13
  • Published : 2019.03.30
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Abstract

Calystegia soldanella is distributed in coastal sand dunes and has high environmental adaptability; it is also known to be effective for anti-oxidant, anti-pyretic, anti-septic, and diuretic action. This study investigated the effect of crude extracts and organic solvent fractions of C. soldanella on MMP-2 and MMP-9 expression, MMP activity, and cell mobility in phorbol-12-myristate-13-acetate (PMA)-induced fibrosarcoma HT-1080 cells. C. soldanella was twice extracted, once with methylene chloride (MC) and once with methanol (MeOH). After the MC and MeOH extracts were combined, their suppressive effects on MMP-2 and MMP-9 expression, MMP enzymatic activity, and gene and protein expression were measured by gelatin zymography, enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and western blot method. Cell mobility for the HT-1080 cells was observed by wound healing assay. The combined crude extracts showed a significant suppressive effects on MMP-2 and MMP-9 expression. To explore active inhibitory elements, the combined extracts were fractionated according to polarity into with n-hexane, 85% aqueous methanol, n-butanol, and water. Across these four solvent fractions, MMP-2 and MMP-9 activity and cell mobility in the HT-1080 cells were all strongly inhibited by the n-hexane fraction. These results suggest that C. soldanella extract and organic solvent fractions could be used as potent MMP inhibitors for effective anti-cancer treatments to suppress cancer invasion and metastasis.

갯메꽃은 해안 사구에 분포하며 식물로 높은 환경 적응력을 가지고 있으며 갯메꽃은 항산화, 해열, 살균, 이뇨작용 등에 효과가 있는 것으로 알려져 있다. 본 연구에서는 암전이 과정에서 중요한 역할을 한다고 알려진 matrix metalloproteinases (MMPs)의 일종인 MMP-2와 MMP-9의 활성에 대한 갯메꽃의 억제효과를 methylene chloride (MC) 및 methanol (MeOH) 추출물과 조추출물, 유기용매 분획물을 시료로 하여 인체 섬유육종 HT-1080 세포를 이용하여 ELISA (Enzyme-linked immunosorbent), gelatin zymography, Wound healing assay, RT-PCR, western blot 실험을 통해 조사하였다. 갯메꽃의 MC 및 MeOH 추출물, 조추출물과 4가지 유기용매 분획물이 HT-1080 세포에서 MMP-2와 MMP-9 생성 억제 활성을 나타냄을 확인하였다. 그 중에서도 n-hexane, 85% aq.MeOH 분획물이 전반적으로 MMP-2와 MMP-9에 대한 높은 억제활성을 보였다. 이들 결과로부터 85% aq.MeOH과 n-hexane 분획물에 MMP 억제 활성이 높은 물질이 존재할 것으로 추측되어지며, 갯메꽃 추출물 및 분획물이 암침윤 및 전이를 억제하는 효과적인 항암소재로서의 가능성이 있음을 제시한다. 이에 본 연구팀은 85% aq.MeOH과 n-hexane 분획물에 있는 활성물질을 분리하기 위한 연구를 추가적으로 수행할 계획이다.

Keywords

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Fig. 1. Effect of methylene chloride (MC) and methanol (MeOH) extracts from C. soldanella on cell cytotoxicity (A) and the production of MMP-2 (B) and MMP-9 (C) in PMA-stimulated HT-1080 human fibrosarcoma cells.

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Fig. 2. Effect of crude extracts from C. soldanella on cell cytotoxicity (A), MMPs expression tested by gelatin zymography (B) and mRNA (C) and protein (D) expression in PMA- stimulated HT-1080 human fibrosarcoma cells.

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Fig. 3. Effect of solvent-partitioned fractions from C. soldanella on cell cytotoxicity (A) and the production of MMP-2 (B) and MMP-9 (C) in PMA-stimulated HT-1080 human fibrosarcoma cells.

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Fig. 4. Effect of solvent-partitioned fractions from C. soldanella on cell motility and invasion by wound migration assay (A) and gelatin zymography (B) in HT-1080 human fibrosarcoma cells.

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Fig. 5. Effect of solvent-partitioned fractions from C. soldanella on MMPs expression in mRNA (A) and protein (B) levels in HT-1080 human fibrosarcoma cells.

Table 1. RT-PCR primer sequence

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References

  1. Ahn, N. R., Ko, J. M. and Cha, H. C. 2012. Comparison of flavonoid profiles between leaves and stems of Calystegia soldanella and Calystegia japonica. Am. J. Plant Sci. 3, 1073-1076. https://doi.org/10.4236/ajps.2012.38128
  2. Brown, P. D., Bioxidge, R. E., Anderson, E. and Howll, A. 1993. Expression of activated gelatinase in human invasive breast carcinoma. Clin. Exp. Metastasis 11, 183-189. https://doi.org/10.1007/BF00114976
  3. Chambers, A. F. and Matrisian, L. M. 1997. Changing views of the role of matrix metalloproteinases in metastasis. J. Natl. Cancer Inst. 89, 1260-1270. https://doi.org/10.1093/jnci/89.17.1260
  4. Gentile, E. and Liuzzi, G. M. 2017. Marine pharmacology: therapeutic targeting of matrix metalloproteinases in neuroinflammation. Drug Discov. Today 22, 299-313. https://doi.org/10.1016/j.drudis.2016.09.023
  5. Gialeli, C., Theocharis, A. D. and Karamanos, N. K. 2011. Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. FEBS J. 278, 16-27. https://doi.org/10.1111/j.1742-4658.2010.07919.x
  6. John, A. and Tuszynski, G. 2001. The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathol. Oncol. Res. 7, 14-23. https://doi.org/10.1007/BF03032599
  7. Kato, Y., Yamashita, T. and Ishikawa, M. 2002. Relationship between expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 and invasion ability of cervical cancer cells. Oncol. Rep. 9, 565-569.
  8. Kim, E. K., Yun, S. J., Do, K. H., Kim, M. S., Cho, M., Suh, D. S., Kim, C. D., Kim, J. H., Birnbaum, M. J. and Bae, S. S. 2008. Lysophosphatidic acid induces cell migration through the selective activation of Akt1. Exp. Mol. Med. 40, 445-452. https://doi.org/10.3858/emm.2008.40.4.445
  9. Lee, J. I., Kim, I. H. and Nam, T. J. 2017. Crude extract and solvent fractions of Calystegia soldanella induce G1 and S phase arrest of the cell cycle in HepG2 cells. Int. J. Oncol. 50, 414-420. https://doi.org/10.3892/ijo.2017.3836
  10. Mook, O. R., Frederiks, W. M. and Van Noorden, C. J. 2004. The role of gelatinases in colorectal cancer progression and metastasis. Biochim. Biophys. Acta Rev. Cancer 1705, 69-89. https://doi.org/10.1016/j.bbcan.2004.09.006
  11. Pavlovic, S., Du, B., Sakamoto, K., Khan, K. M., Natarajan, C., Breyer, R. M., Dannenberg, A. J. and Falcone, D. J. 2006. Targeting prostaglandin E2 receptors as an alternative strategy to block cyclooxygenase-2-dependent extracellular matrix-induced matrix metalloproteinase-9 expression by macrophages. J. Biol. Chem. 281, 3321-3328. https://doi.org/10.1074/jbc.M506846200
  12. Pezzuto, J. M. 1997. Plant-derived anticancer agents. Biochem. Pharmacol. 53, 121-133. https://doi.org/10.1016/S0006-2952(96)00654-5
  13. Ramos-DeSimone, N., Hahn-Dantona, E., Sipley, J., Nagase, H., French, D. L. and Quigley, J. P. 1999. Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/ stromelysin-1 cascade enhances tumor cell invasion. J. Biol. Chem. 274, 13066-13076. https://doi.org/10.1074/jbc.274.19.13066
  14. Siegel, R. L., Miller, K. D. and Jemal, A. 2017. Cancer statistics 2017. CA Cancer J. Clin. 67, 7-30. https://doi.org/10.3322/caac.21387
  15. Soreide, K., Janssen, E. A., Korner, H. and Baak, J. P. 2006. Trypsin in colorectal cancer: molecular biological mechanisms of proliferation, invasion, and metastasis. J. Pathol. 209, 147-156. https://doi.org/10.1002/path.1999
  16. Spano, C., Bruno, M. and Bottega, S. 2013. Calystegia soldanella: dune versus laboratory plants to highlight key adaptive physiological trait. Acta Physiol. Plant. 35, 1329-1336 https://doi.org/10.1007/s11738-012-1173-x
  17. Stetler-Stevenson, W. G. 1990. Type IV collagenases in tumor invasion and metastasis. Cancer Metastasis Rev. 9, 289-303. https://doi.org/10.1007/BF00049520
  18. Tori, M., Ohara, Y., Nakashima, K. and Sono, M. 2000. Caffeic and courmaric acid esters from Calystegia soldanella. Fitoterapia 71, 353-359. https://doi.org/10.1016/S0367-326X(99)00174-4
  19. Vanmeter, T. E., Rooprai, H. K., Kibble, M. M., Fillmore, H. L., Broaddus, W. C. and Pilkington, G. J. 2001. The role of matrix metalloproteinase genes in glioma invasion: co-dependent and interactive proteolysis. J. Neurooncol. 53, 213-235. https://doi.org/10.1023/A:1012280925031
  20. Yang, Y. H. 2002. The relationship of symptoms of side effects, fatigue and quality of life in stomach cancer patients receiving chemotherapy. J. Korea Acad. Nurs. 14, 205-212.
  21. Zeng, Z. S., Cohen, A. M. and Guillem, J. G. 1999. Loss of basement membrane type IV collagen is associated with increased expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) during human colorectal tumorigenesis. Carcinogenesis 20, 749-755. https://doi.org/10.1093/carcin/20.5.749