Analysis of Differentially Expressed Genes by Allicin in Human Colorectal Cancer Cell

마늘성분 Allicin에 의해 차별적으로 발현되는 유전자군의 발현 분석

  • Kim, Kyung-Ho (Devision of immunopharmacology, School of Pharmacy, Sungkyunkwan University) ;
  • Kim, Min-Jung (Department of Biological Sciences, Andong National University) ;
  • Kim, Jong-Sik (Department of Biological Sciences, Andong National University) ;
  • Pyo, Suhk-Neung (Devision of immunopharmacology, School of Pharmacy, Sungkyunkwan University) ;
  • Kim, Byung-Oh (Department of Applied Biology, Kyungpook National University)
  • Received : 2010.09.14
  • Accepted : 2010.10.06
  • Published : 2010.12.28

Abstract

We investigated anti-cancer and anti-proliferative activity of allicin and analyzed global gene expression changes by allicin treatment in human colorectal HCT116 cells. As a result, allicin decreased cell viabilities in a dose and time-dependent manner and induced apoptosis. Oligo DNA microarray analysis, we found that 7,840 genes were up-regulated more than 2-folds, whereas 10,010 genes were down-regulated more than 2-folds by $50\;{\mu}M$ allicin treatment. To confirm specific gene expressions, we performed RT-PCR. Consistent with the results of DNA microarray analysis, allicin dramatically induced ATF3 and NAG1 gene expression. Interestingly, NAG-1 protein expression was dependent on p53 presence. Taken together, our present results increase the knowledge of the molecular mechanism of anti-cancer and anti-proliferative activity mediated by allciin in human colorectal cancer cell.

본 연구에서는 마늘성분 allicin에 의한 항암 기전 및 화학적 암 예방법의 분자 생물학적 기전을 이해하기 위한 연구의 일환으로, 마늘성분 중의 하나인 allicin이 인체 대장암 세포주 HCT116의 증식에 미치는 영향을 확인하였다. allicin의 처리에 따라 세포사멸이 나타나며 생존율이 감소함을 확인하였다. 또한 oligo DNA microarray 실험을 통하여 allicin에 의해 발현되는 유전자 중 발현양의 차이를 보인 유전자중 2배 이상 up-regulation된 유전자는 7,840개, 2배 이상 down-regulation 된 유전자는 10,010개로 각각 분류 되었다. 이 중, ATF3유전자의 발현을 확인하였고, 또 다른 항암유전자인 NAG-1 유전자의 발현을 RT-PCR로 확인하였다. 그 결과, allicin처리에 의해 항암유전자인 ATF3의 발현과 NAG1 유전자의 발현이 증가함을 확인하였다. 또한 p53 HCT116 세포주를 이용하여 allicin에 의한 NAG1 단백질의 발현을 확인한 결과, allicin은 p53 유전자 의존적으로 NAG-1 단백질을 발현함을 확인하였다. 결론적으로 allicin은 세포주기나 세포사멸에 관련된 많은 유전자들의 발현 변화를 유도함으로써 암세포 성장억제 활성을 갖는 것으로 사료된다.

Keywords

References

  1. Agarwal, K. C. 1996. Therapeutic actions of garlic constituents. Med. Res. Rev. 16: 111-124. https://doi.org/10.1002/(SICI)1098-1128(199601)16:1<111::AID-MED4>3.0.CO;2-5
  2. Aggarwal, B. B., S. Banerjee, U. Bharadwaj, B. Sung, S. Shishodia, and G. Sethi. 2007. Curcumin induces the degradation of cyclin E expression through ubiquitin-dependent pathway and up-regulates cylin-dependent kinase inhibitors p21 and p27 in multiple human tumor cell lines. Biochem. Pharmacol. 73: 1024-1032. https://doi.org/10.1016/j.bcp.2006.12.010
  3. Aggarwal, B. B. and S. Shishodia. 2006. Molecular targets of dietary agents for prevention and therapy of cancer. Biochem. Pharmacol. 14: 1397-1421.
  4. Ahmed, N., L. Laverick, J. Sammons, H. Zhang, D. J. Maslin, and H. T. Hassan. 2001. Ajoene, a garlic-derived natural compound, enhances chemotherapy-induced apoptosis in human myeloid leukaemia CD34-positive resistant cells. Anticancer Res. 21: 3519-3523.
  5. Arditti, F. D. A. Rabinkov, T. Miron, Y. Reisner, A. Berrebi, M. Wilchek and D. Mirelman. 2005. Apoptotic killing of Bchronic lymphocytic leukemia tumor cells by allicin generated in situ using a rituximab-alliinase conjugate. Mol. Cancer Ther. 4: 325-331.
  6. Baek, S. J., J. S. Kim, F. R. Jackson, T. E. Eling, M. F. McEntee, and S. H. Lee. 2004. Epicatechin gallate-induced expression of NAG-1 is associated with growth inhibition and apoptosis in colon cancer cells. Carcinogenesis. 25: 2425-2432. https://doi.org/10.1093/carcin/bgh255
  7. Baek, S. J., K. S. Kim, J. B. Nixon, L. C. Wilson, and T. E. Eling. 2001. Cyclooxygenase inhibitors regulate the expression of a TGF-beta superfamily member that has proapoptotic and antitumorigenic activities. Mol. Pharmacol. 59: 901-908.
  8. Baek, S. J., L. C. Wilson, and T. E. Eling. 2002. Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53. Carcinogenesis. 23: 425-434. https://doi.org/10.1093/carcin/23.3.425
  9. Baek, S. J., R. Okazaki, S. H. Lee, J. Martinez, J. S. Kim, K. Yamaguchi, Y. Mishina, D. W. Martin, A. Shoieb, M. F. McEntee, and T. E. Eling. 2006. Nonsteroidal anti-inflammatory drug-activated gene-1 over expression in transgenic mice suppresses intestinal neoplasia. Gastroenterology. 131: 1553-1560. https://doi.org/10.1053/j.gastro.2006.09.015
  10. Balasenthil, S. K. S. Rao, and S. Nagini. 2002. Altered cytokeratin expression during chemoprevention of experimental hamster buccal pouch carcinogenesis by garlic. J. Oral. Pathol. Med. 31: 142-146. https://doi.org/10.1034/j.1600-0714.2002.310303.x
  11. Belloir, C. V. Singh, C. Daurat, M. H. Siess, and A. M. Le Bon. 2006. Protective effects of garlic sulfur compounds against DNA damage induced by direct- and indirect-acting genotoxic agents in HepG2 cells. Food Chem. Toxicol. 44: 827-834. https://doi.org/10.1016/j.fct.2005.11.005
  12. Dorai, T and Aggarwal, B. B. 2004. Role of chemopreventive agents in cancer therapy. Cancer Lett. 25: 129-140.
  13. Ghodrati Azadi, H. S. H. Ghaffari, G. H. Riazi, S. Ahmadian, and F. Vahedi. 2008. Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines. Cytotechnology. 56: 179-185. https://doi.org/10.1007/s10616-008-9145-0
  14. Gu, Y., C. F. Zhu, H. Iwamoto, and J. S. Chen. 2005. Genistein inhibits invasive potential of human hepatomacellular carcinoma by altering cell cycle, apoptosis, and angiogenesis. World J. Gastroenterol. 11: 6512-6517.
  15. Johnson, I. T. 2007. Phytochemicals and cancer. Proc. Nutr. Soc. 66: 207-215. https://doi.org/10.1017/S0029665107005459
  16. Kim, H. Y. and E. J. Yang. 1993. A study on dietary factors related to the incidence of stomach cancer and colon cancer in Korea. Kor. J. Nutr. 26: 603-614.
  17. Lee, S. H., J. S. Kim, K. Yamaguchi, T. E. Eling, and S. J. Baek. 2005. Indole-3-carbinol and 3,3'-diindolylmethane induce expression of NAG-1 in a p53-independent manner. Biochem. Biophys. Res. Commun. 328: 63-69. https://doi.org/10.1016/j.bbrc.2004.12.138
  18. Lee, S. J., J. H. Shin, M. J. Kang, W. J. Jung, J. H. Ryu, R. J. Kim, and N. J. Sung. 2010. Antioxidants activity of aged red garlic. J. Life Sci. 20: 775-781. https://doi.org/10.5352/JLS.2010.20.5.775
  19. Li. Y., J. Liu, X. Liu, K. XIng, Y. Wang, F. Li, and L. Yao. 2006. Resveratrol-induced cell inhibition of growth and apoptosis in MCF7 human breast cancer cells are associated with modulation of phosphorylated AKT and caspase-9. Appl. Biochem. Biotechnol. 135: 181-192. https://doi.org/10.1385/ABAB:135:3:181
  20. Miron, T. M. Mironchik, D. Mirelman, M. Wilchek, and A. Rabinkov. 2003. Inhibition of tumor growth by a novel approach: in situ allicin generation using targeted alliinase delivery. Mol. Cancer Ther. 2: 1295-301.
  21. Munchberg, U. A. Anwar, S. Mecklenburg, and C. Jacob. 2007. Polysulfides as biologically active ingredients of garlic. Org. Biomol. Chem. 5: 1505-1518. https://doi.org/10.1039/b703832a
  22. Oommen, S. R. J. Anto, G. Srinivas, and D. Karunagaran. 2004. Allicin (from garlic) induces caspase-mediated apoptosis in cancer cells. Eur. J. Pharmacol. 485: 97-103. https://doi.org/10.1016/j.ejphar.2003.11.059
  23. Rahman, K. W., Y. Li, Z. Wang, S. H. Sarkar, and F. H. Sarkar. 2006. Gene expression profiling reavealed survivin as a target of 3,3'-diindolylmethane-induced cell growth inhibition and apoptosis in breast cancer cell. Cancer Res. 66: 4952-4960. https://doi.org/10.1158/0008-5472.CAN-05-3918
  24. Son, E. W. S. J. Mo, D. K. Rhee, and S. Pyo. 2006. Inhibition of ICAM-1 expression by garlic component, allicin, in gamma-irradiated human vascular endothelial cells via downregulation of the JNK signaling pathway. Int. Immunopharmacol. 6: 1788-1795. https://doi.org/10.1016/j.intimp.2006.07.021
  25. Wilson, L. C. S. J. Baek, A. Call, and T. E. Eling. 2003. Non-steroidal anti-inflammatory drug-activated gene (NAG- 1) is induced by genistein through the expression of p53 in colorectal cancer cells. Int. J. Cancer. 105: 747-753. https://doi.org/10.1002/ijc.11173
  26. Yang, S. T. 2007. Antioxidants activity of extracts of aged black garlic on oxidation of human low density lipoprotein. J. Life Sci. 17: 1330-1335. https://doi.org/10.5352/JLS.2007.17.10.1330