Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
권호 표지
DOI QR코드

DOI QR Code

Anti-Cancer Effect of Ursolic Acid in Melanoma Cell A375SM and A375P

Ursolic acid의 악성 흑색종 세포주 A375SM과 A375P에서의 항암효능

  • Woo, Joong-Seok (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Kim, Na-Won (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Lee, Jin-Gyu (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Kim, Jae-Hyuk (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Lim, Da-Young (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Kang, Shin-Woo (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Kim, Sung-Hyun (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Yoo, Eun-Seon (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Lee, Jae-Han (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Han, So-Hee (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Park, Young-Seok (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Kim, Byeong-Soo (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Kim, Sang-Ki (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Park, Byung-Kwon (Department of Companion and Laboratory Animal Science, Kongju National University) ;
  • Jung, Ji-Youn (Department of Companion and Laboratory Animal Science, Kongju National University)
  • 우중석 (공주대학교 특수동물학과) ;
  • 김나원 (공주대학교 특수동물학과) ;
  • 이진규 (공주대학교 특수동물학과) ;
  • 김재혁 (공주대학교 특수동물학과) ;
  • 임다영 (공주대학교 특수동물학과) ;
  • 강신우 (공주대학교 특수동물학과) ;
  • 김성현 (공주대학교 특수동물학과) ;
  • 유은선 (공주대학교 특수동물학과) ;
  • 이재한 (공주대학교 특수동물학과) ;
  • 한소희 (공주대학교 특수동물학과) ;
  • 박영석 (공주대학교 특수동물학과) ;
  • 김병수 (공주대학교 특수동물학과) ;
  • 김상기 (공주대학교 특수동물학과) ;
  • 박병권 (공주대학교 특수동물학과) ;
  • 정지윤 (공주대학교 특수동물학과)
  • Received : 2018.12.21
  • Accepted : 2019.02.16
  • Published : 2019.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

Ursolic acid is recognized for various effects such as anti-cancer, antioxidant, and anti-inflammatory activity. In this study, we confirmed the anti-cancer effect of ursolic acid on human melanoma cancer cells, A375SM and A375P. Survival rate of the melanoma cells was confirmed by MTT assay and the proliferation rate was confirmed by wound healing assay. The rate of apoptotic bodies was confirmed by DAPI staining, and apoptosis rate was confirmed by flow cytometry. The induction of apoptosis protein was examined by western blotting according to the concentration of ursolic acid in melanoma cells. The survival and proliferation rates of melanoma cells were decreased according to the treatment concentrations of ursolic acid. DAPI staining showed that chromosomal condensation of melanoma cells was increased with increasing concentrations of ursolic acid, and increased apoptosis rate of melanoma cells by ursolic acid was confirmed by flow cytometry. We also confirmed by western blotting that cleaved-PARP and Bax were increased and Bcl-2 was decreased at $12{\mu}M$ concentration of uricolic acid in melanoma cells. This study was carried out at low concentrations of ursolic acid, 0 to $20{\mu}M$, and analyzed 24 h after treatment. As a result of this study, it is thought that ursolic acid has the anti-cancer effect through the regulation of apoptosis-related proteins in melanoma cells A375SM and A375P.

우르솔릭산은 항암, 항산화, 항염증 작용과 같은 다양한 효과를 지니고 있다. 본 연구에서는 우르솔릭산이 인간 흑색종 암세포인 A375SM과 A375P 세포에 항암효과가 있는지 확인하였다. 두 세포의 생존율은 MTT assay를 통하여 확인하였으며 증식률은 Wound healing assay로 확인하였다. 두 세포의 apoptotic body와 apoptosis 비율의 확인을 위한 DAPI 염색과 유세포 분석을 진행하였다. 그리고 웨스턴 블로팅을 통하여 흑색종 세포의 우르솔릭산의 농도에 따른 apoptosis 단백질의 유도를 조사하였다. 우르솔릭산의 처리 농도에 따라 흑색종 세포의 생존율 감소와 증식률 감소를 확인하였다. DAPI 염색을 통하여 우르솔릭산의 농도가 증가함에 따라 흑색종 세포의 염색체 응축이 농도 의존적으로 증가하였고, 유세포 분석을 통하여 우르솔릭산에 대하여 농도 의존적으로 흑색종 세포의 apoptosis 비율의 증가를 확인하였다. 그리고 웨스턴 블로팅을 통해 흑색종 세포 A375SM과 A375P의 우르솔릭산 $12{\mu}M$ 농도에서 cleaved-PARP와 Bax의 증가와 Bcl-2의 감소를 확인하였다. 본 연구는 우르솔릭산의 농도를 0 에서 $20{\mu}M$ 수준의 저농도에서 진행하였으며, 물질 처리 후 24 시간 뒤 결과를 가지고 분석하였다. 본 연구의 결과로 보아 우르솔릭산은 흑색종 세포 A375SM과 A375P에서 apoptosis 관련 단백질들의 조절을 통해 항암효과를 일으키는 것으로 사료된다.

Keywords

References

  1. Wang H.M., Chen C.Y., Chen C.Y., Ho M.L., Chou Y.T., Chang H.C., Lee C.H., Wang C.Z., Chu I.M.: (-)-N-Formylanonaine from Michelia alba as a human tyrosinase inhibitor and antioxidant. Bioorg. Med. Chem., 18, 5241-5247 (2010). https://doi.org/10.1016/j.bmc.2010.05.045
  2. Briganti S., Picardo M.: Antioxidant activity, lipid peroxidation and skin diseases. What's new. J. Eur. Acad. Dermatol. Venereol., 17, 663-669 (2003). https://doi.org/10.1046/j.1468-3083.2003.00751.x
  3. Picardo M., Grammatico P., Roccella F., Roccella M., Grandinetti M., Del Porto G., Passi S.: Imbalance in the antioxidant pool in melanoma cells and normal melanocytes from patients with melanoma. J. Invest. Dermatol., 107, 322-336 (1996). https://doi.org/10.1111/1523-1747.ep12363163
  4. Lens M.B., Dawes M.: Global perspectives of contemporary epidemiological trends of cutaneous malignant melanoma. British Journal of Dermatology, 150, 179-185 (2004). https://doi.org/10.1111/j.1365-2133.2004.05708.x
  5. Rigel D.S., Russak J., Friedman R.: The evolution of melanoma diagnosis: 25 years beyond the ABCDs. CA: A Cancer Journal for Clinicians, 60, 301-316 (2010). https://doi.org/10.3322/caac.20074
  6. Health Insurance Review & Assessment Service. Available from http://opendata.hira.or.kr/op/opc/olapMfrnIntrsIlnsInfo.do
  7. Lee J.W.: The apoptotic effect of allicin in human melanoma cell line A375.S2. Doctoral thesis. Pusan University, Busan, Korea. (2015).
  8. Bhatia S., Tykodi S.S., Thompson J.A.: Treatment of metastatic melanoma: An overview. Oncology -Williston Park Then Huntington-, 23, 488-495 (2009).
  9. Sivastava S.K., Singh S.V.: Cell cycle arrest, apoptosis induction and inhibition of nuclear factor kappa B activation in anti-proliferative activity of benzyl isothiocyanate against human pancreatic cancer cells. Carcinogenesis, 25, 1701-1709 (2004). https://doi.org/10.1093/carcin/bgh179
  10. Ma Y.S., Hsu S.C., Weng S.W., Yu C.C., Yang J.S., Lai K.C., Lin J.G., Chung J.G.: Crude extract of Rheum palmatum L induced cell death in LS1034 human colon cancer cells acts through the caspase-dependent and -independent pathways. Environ. Toxicol., 29, 969-980 (2014). https://doi.org/10.1002/tox.21827
  11. Koo H.N., Jeong H.J., Choi I.Y., An H.J., Moon P.D., Kim S.J., Jee S.Y., Um J.Y., Hong S.H., Shin S.S., Yang D.C., Seo Y.S., Kim H.M.: Mountain grown ginseng induces apoptosis in HL-60 cells and its mechanism have little relation with TNF-alpha production. The American journal of Chinese medicine, 35, 169-182 (2007). https://doi.org/10.1142/S0192415X07004710
  12. Ha D.W.: Effects of terpenoids and organosulfur-rich plant extracts on rumen methane emission. Master Thesis. Gyeongsang University, Gyeongsangnam-do, Korea. (2013).
  13. Ikeda Y., Murakami A., Ohigashi H.: Ursolic acid: An anti- and pro-inflammatory triterpenoid. Molecular Nutrition & Food Research, 52, 26-42 (2008). https://doi.org/10.1002/mnfr.200700389
  14. Forsyth N.R., Musio A., Vezzoni P., Simpson A.H., Noble B.S., McWhir J.: Physiologic oxygen enhances human embryonic stem cell clonal recovery and reduces chromosomal abnormalities. Cloning and Stem Cells, 8, 16-23 (2006). https://doi.org/10.1089/clo.2006.8.16
  15. Checker R., Sandur S.K., Sharma D., Patwardhan R.S., Jayakumar S., Kohli V., Sethi G., Aggarwal B.B., Sainis K.B.: Potent anti-inflammatory activity of ursolic acid, a triterpenoid antioxidant, is mediated through suppression of NF-${\kappa}B$, AP-1 and NF-AT. PLoS One, 7, e31318 (2012). https://doi.org/10.1371/journal.pone.0031318
  16. Prasad S., Yadav V.R., Kannappan R., Aggarwal B.B.: Ursolic acid, a pentacyclin triterpene, potentiates TRAIL-induced apoptosis through p53-independent up-regulation of death receptors. Evidence for the role of reactive oxygen species and jnk. The journal of biological chemistry, 291, 16924 (2016). https://doi.org/10.1074/jbc.A110.183699
  17. Liu J.: Pharmacology of oleanolic acid and ursolic acid. Journal of ethnopharmacology, 49, 57-68 (1995). https://doi.org/10.1016/0378-8741(95)90032-2
  18. Mezzetti T., Orzalesi G., Bellavita V.: Chemistry of ursolic acid. Planta Medica, 20, 244-252 (1971). https://doi.org/10.1055/s-0028-1099700
  19. Shan J.Z., Xuan Y.Y., Zheng S., Dong Q., Zhang S.Z.: Ursolic acid inhibits proliferation and induces apoptosis of HT-29 colon cancer cells by inhibiting the EGFR/MAPK pathway. J. Zhejiang University. Science. B., 10, 668-678 (2009). https://doi.org/10.1631/jzus.B0920149
  20. Chadalapaka G., Jutooru I., McAlees A., Stefanac T., Safe S.: Structure-dependent inhibition of bladder and pancreatic cancer cell growth by 2-substituted glycyrrhetinic and ursolic acid derivatives. Bioorganic & Medicinal Chemistry letters, 18, 2633-2639 (2008). https://doi.org/10.1016/j.bmcl.2008.03.031
  21. Shanmugam M.K., Ong T.H., Kumar A.P., Lun C.K., Ho P.C., Wong P.T., Hui K.M., Sethi G.: Ursolic acid inhibits the initiation, progression of prostate cancer and prolongs the survival of TRAMP mice by modulating pro-inflammatory pathways. PLoS One, 7, e32476 (2012). https://doi.org/10.1371/journal.pone.0032476
  22. Wang X., Zhang F., Yang L., Mei Y., Long H., Zhang X., Zhang J., Qimuge-Suyila, Su X.: Ursolic acid inhibits proliferation and induces apoptosis of cancer cells in vitro and in vivo. Journal of biomedicine & biotechnology, 2011:419343 (2011).
  23. Li J., Guo W.J., Yang Q.Y.: Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15. World journal of gastroenterology, 8, 493-495 (2002). https://doi.org/10.3748/wjg.v8.i3.493
  24. Ray N.: Ursolic acid inhibits cell growth in esophageal squamous cell carcinoma by inducing autophagy. Master Thesis. Chonbuk University, Korea. (2016).
  25. Choi B.M., Park R., Pae H.O., Yoo J.C., Kim Y.C., Jun C.D., Jung B.H., Oh G.S., So H.S., Kim Y.M., Chung H.T.: Cyclic adenosine monophosphate inhibits ursolic acid-induced apoptosis via activation of protein kinase A in human leukaemic HL-60 cells. Pharmacology & toxicology, 86, 53-58 (2000). https://doi.org/10.1034/j.1600-0773.2000.d01-10.x
  26. Shan J., Xuan Y., Zhang Q., Zhu C., Liu Z., Zhang S.: Ursolic acid synergistically enhances the therapeutic effects of oxaliplatin in colorectal cancer. Protein Cell, 7, 571-585 (2016). https://doi.org/10.1007/s13238-016-0295-0
  27. Kashyap D., Tuli H.S., Sharma A.K.: Ursolic acid (UA): A metabolite with promising therapeutic potential. Life Sciences, 146, 201-203 (2016). https://doi.org/10.1016/j.lfs.2016.01.017