Sains Malaysiana 52(6)(2023): 1811-1820

http://doi.org/10.17576/jsm-2023-5206-16

The Effect of Lunasin on Inhibition of KI67, BCL-2 and C-MYC Expression in Azoxymethane and Dextran Sodium Sulfate Induced Mice Colon  

(Kesan Lunasin terhadap Perencatan Ekspresi KI67, BCL-2 dan C-MYC dalam Kolon Tikus Terinduksi Azoksimetana dan Dektran Natrium Sulfat)

KUSMARDI KUSMARDI^1,2,3,4,*, AHMAT REDIANSYA PUTRA⁵ & RIA KODARIAH¹

¹Department of Anatomical pathology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

²Drug Development Research Cluster, Indonesian Medical Education and Research Institute, Universitas Indonesia, Jakarta, Indonesia

³Human Cancer Research Cluster, Indonesian Medical Education and Research Institute, Universitas Indonesia, Jakarta, Indonesia

⁴Doctoral Program for Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

⁵Graduate Student, Master’s Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jalan Salemba Raya No 6, Jakarta, Indonesia

Received: 26 July 2022/Accepted: 2 June 2023

Abstract

Treatment of cancer using medicinal-plant based has been important due to minimal side effects, high efficiency and low cost. Lunasin from soybean is known as potential chemopreventive agent. This study aimed to study and investigate the proteins involved in the mechanisms of action of lunasin underlie its chemopreventive effects in Azoxymethane (AOM) and Dextran Sodium Sulfate (DSS) induced mice. A total 30 BLAB/c mice were separated into six groups. In five of the groups - a negative control group, positive control group, and three intervention groups - carcinogenesis was induced with AOM and DSS; the sixth group received no interventions. Lunasin were given in different doses of Low Dose Lunasin (75 mg/kgBW), Moderate Dose Lunasin (150 mg/kgBW), and High Dose Lunasin (200 mg/kgBW) to intervention groups. Immunohistochemistry was conducted to measure Ki67, C-myc, and Bcl-2 expressions from the distal colons of mice that had been sacrificed. The samples were microscopically assessed and photographed, and cell counts were performed using the Image J application. Further, the H-score method was used to quantify of Ki67, C-myc and Bcl-2 expressions. The results of this show that there is significant differences between the negative control and the intervention groups were found at the 75 mg/kgBW and 150 mg/kgBW (p < 0.05) lunasin dosage levels. This demonstrates that Lunasin inhibits proliferation and induces apoptosis in the colon mice induced by AOM and DSS.

Keywords: AOM; Bcl-2; C-myc; DSS; Ki67

Abstrak

Rawatan kanser berasaskan tumbuhan ubatan adalah penting kerana kesan sampingan yang minimum, kecekapan tinggi dan kos yang rendah. Lunasin daripada kacang soya dikenali sebagai agen kemohalang yang berpotensi. Penyelidikan ini bertujuan untuk mengkaji protein yang terlibat dalam mekanisme tindakan lunasin yang mendasari kesan kemohalangnya pada tikus yang disebabkan oleh Azoksimetana (AOM) dan Dektran Natrium Sulfat (DSS). Sebanyak 30 tikus BLAB/c dipisahkan kepada enam kumpulan. Dalam lima daripada kumpulan kawalan negatif, kumpulan kawalan positif dan tiga kumpulan intervensi-karsinogenesis telah diinduksi dengan AOM dan DSS; kumpulan keenam tidak menerima campur tangan. Lunasin diberikan dalam dos berbeza Lunasin Dos Rendah (75 mg/kgBW), Lunasin Dos Sederhana (150 mg/kgBW) dan Lunasin Dos Tinggi (200 mg/kgBW) kepada kumpulan intervensi. Imunohistokimia telah dijalankan untuk mengukur ekspresi Ki67, C-myc dan Bcl-2 daripada kolon distal tikus yang telah dikorbankan. Sampel telah dinilai secara mikroskopik dan difoto dan kiraan sel dilakukan menggunakan aplikasi Image J. Selanjutnya, kaedah H-skor digunakan untuk mengukur ungkapan Ki67, C-myc dan Bcl-2. Keputusan ini menunjukkan terdapat perbezaan yang signifikan antara kawalan negatif dan kumpulan intervensi didapati pada tahap dos lunasin 75 mg/kgBW dan 150 mg/kgBW (p < 0.05). Ini menunjukkan bahawa Lunasin menghalang percambahan dan mendorong apoptosis dalam tikus kolon yang disebabkan oleh AOM dan DSS.

Kata kunci: AOM; Bcl-2; C-myc; DSS; Ki67

REFERENCES

Amalia, A.W., Kusmardi, Elya, B. & Arsianti, A. 2017. Inhibition of carcinogenesis by seed and soybean meal extract in colon of mice: Apoptosis and dysplasia. Asian Journal of Pharmaceutical and Clinical Research 10(4): 123-128.

American, C.S. 2017. Colorectal Cancer Facts & Figures 2017-2019. Atlanta: American Cancer Society.

Bagher, M., Hesar, A.R., Sarabi, P.Z., Rahimi, H.R., Maryam, B. & Ghasem, F. 2018. Antiproliferative effect of aspirin on colorectal cancer cell line. Iran Journal of Toxicology 12(5): 3-6.

Dia, V.P. & Mejia, E.G. 2011. Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to a5b1 integrin and suppresses. Cancer Letters 313: 167-180.

He, W.L., Weng, X.T., Wang, J.L., Lin, Y.K., Liu, T.W. & Zhou, Q.Y. 2018. Association between c-Myc and colorectal cancer prognosis: A meta-analysis. Frontier in Physiology 9: 1-9.

Hirano, T., Hirayama, D., Wagatsuma, K., Yamakawa, T., Yokoyama, Y. & Hiroshi, N. 2020. Immunological mechanisms in inflammation-Associated colon carcinogenesis. International Journal of Molecular Science 21(9): 3062.

Hsieh, C.C., Hernández-Ledesma, B., Jeong, H.J., Park, J.H. & De Lumen, B.O. 2010. Complementary roles in cancer prevention: Protease inhibitor makes the cancer preventive peptide Lunasin bioavailable. PLoS ONE 5(1): e8890.

Ibadawy, M., Usui, T., Yamawaki, H. & Sasaki, K. 2019. Emerging roles of C-myc in cancer stem cell-related signaling and resistance to cancer chemotherapy: A potential therapeutic target against colorectal cancer. International Journal of Molecular Science 20(9): 2340.

Jia, S., Zhang, S., Yuan, H. & Chen, N. 2015. Lunasin inhibits cell proliferation via apoptosis and reduces the production of proinflammatory cytokines in cultured rheumatoid arthritis synovial fibroblasts. Biomedic Research International 2015: 346839.

Jiang, Q., Pan, Y.U., Cheng, Y., Li, H., Liu, D. & Li, H. 2016. Lunasin suppresses the migration and invasion of breast cancer cells by inhibiting matrix metalloproteinase-2/-9 via the FAK/Akt/ERK and NF-κB signaling pathways. Oncology Reports 10: 253-262.

Kim, E.R. & Chang, D.K. 2014. Colorectal cancer in inflammatory bowel disease: The risk, pathogenesis, prevention and diagnosis. World Journal of Gastroenterology 20(29): 9872-9881.

Kumar, V., Abbas, K.A. & Aster, C.J. 2013. Robbin Basic Pathology. 9th ed. Singapore: Elsevier Pte Ltd.

Kusmardi, Karenina, V., Estuningtyas, A. & Tedjo, A. 2019a. Effect of lunasin-rich soybean extract upon TNF-α expression on colon epithelial cells of mice induced by azoxymethane/dextrane sodium sulfate. International Journal of Applied Pharmaceutics 11(6): 12-16.

Kusmardi, Karenina, V., Estuningtyas, A. & Tedjo, A. 2019b. Inhibition of COX-2 expression by lunasin-rich soybean extract on colorectal cancer. International Journal of Applied Pharmaceutics 11(6): 116-121.

Kusmardi, Rosa, T., Tarigan, S., Estuningtyas, A. & Tedjo, A. 2019c. Effect of lunasin-enriched soy extract on histone deacetylase expression in distal colon epithelial cells from AOM/DSS-induced mice. International Journal of Applied Pharmaceutics 11(6): 111-115.

Kusmardi, Nessa, N., Estuningtyas, A. & Tedjo, A. 2018a. The effect of lunasin from Indonesian soybean extract on histopatologic examination and COX-2 expression in dextran sodium sulfate-induced mice colon. International Journal of Applied Pharmaceutics 10(6): 154-162.

Kusmardi, Nessa, N., Estuningtyas, A., Tedjo, A. & Wuyung P.E. 2018b. The effect of lunasin from Indonesian soybean extract on inducible nitric oxide synthase and β-catenin expression in dextrane sodium sulfate-induced mice colon. International Journal of Applied Pharmaceutics 11(1): 416-420.

Lin, R., Piao, M., Song, Y. & Liu, C. 2020. Quercetin suppresses AOM/DSS-induced colon carcinogenesis through its anti-inflammation effects in mice. Journal of Immunology Research 2020: 9242601.

Lu, Q.L., Abel, P., Foster, C.S. & Lalani, E.N. 1996. Bcl-2: Role in epithelial differentiation and oncogenesis. Human Pathology 27(2): 102-110.

Mantovani, A., Allavena, P., Sica, A. & Balkwill, F. 2008. Cancer-related inflammation. Nature 454(7203) 436-44.

Menon, S.S., Guruvayoorappan, C., Sakthivel, K.M. & Rasmi, R.R. 2019. Ki-67 protein as a tumor proliferation marker. Clinica Chimita Acta491: 39-45.

Meteoglu, I., Erdogdu, I.H., Tuncyurek, P., Coskun, A., Culhaci, N. & Erkus, M. 2018. Nuclear factor kappa B, matrix metalloproteinase-1, p53, and Ki-67 expressions in the primary tumors and the lymph node metastases of colorectal cancer cases. Gastroenterology Research Practice 2018: 945392.

Mohan, S., Abdelwahab, S.I., Kamalidehghan, B., Syam, S., May, K.S. & Harmal, N.S.M. 2012. Involvement of NF-κB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A. Phytomedicine 19(11): 1007-1015.

Pabona, M.P., Dave, B., Su, Y., Montales, M.T.E., De Lumen, B.O. & De Mejia, E.G. 2013. The soybean peptide lunasin promotes apoptosis of mammary epithelial cells via induction of tumor suppressor PTEN: Similarities and distinct actions from soy isoflavone genistein. Genes and Nutrition 8(1): 79-90.

Patel, M., Horgan, P.G., Mcmillan, D.C. & Edwards, J. 2018. NF-κB pathways in the development and progression of colorectal cancer. Translational Research 197: 43-56.

Rawla, P., Sunkara, T. & Barsouk, A. 2019. Epidemiology of colorectal cancer: Incidence, mortality, survival, and risk factors. Gastroenterol. Rev. 14: 89-103.

Robertis, M.D., Massi, E., Poeta, M.L., Carottu, S., Morini, S. & Cecchetelli, L. 2011. The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies. Journal of Carcinogenesis 10: 9.

Scherr, A-L. 2018. The Role of Anti-Apoptotic Bcl-2 Proteins for Colorectal Cancer Development and Progression. Germany: Ruperto-Carola University of Heidelberg.

Suzuki, R., Kohno, H. & Sugie, S. 2006. Strain differences in the susceptibility to azoxymethane and dextran sodium sulfate-induced colon carcinogenesis in mice. Carcinogenesis 27: 162-169.

Tanaka, T., Kohno, H. & Suzuki, R. 2003. A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate. Cancer Science 94: 965-973.

Tomlinson, I.P.M., Hanby, A.M., Yao, T., Bodmer, W.F. & Talbot, I.C. 1996. BcI-2 expression in colorectal tumors: Evidence of different pathways in sporadic and ulcerative-colitis-associated carcinomas. American Journal of Pathology 149: 1719-1726.

Wan, X., Liu, H., Sun, Y., Zhang, J., Chen, X. & Chen, N. 2017. Lunasin: A promising polypeptide for the prevention and treatment of cancer (Review). Oncology Letters 13(6): 3997-4001.

*Corresponding author; email: kusmardis@gmail.com