Interrelations of Apoptotic and Cellular Senescence Genes Methylation in Inflammatory Bowel Disease Subtypes and Colorectal Carcinoma in Egyptians Patients

  • Ragaa H. Salama
  • Zain El-Abdeen A. Sayed
  • Ahmed M. Ashmawy
  • Wael A. Elsewify
  • Ghada M. EzzatEmail author
  • Mahmoud A. Mahmoud
  • Aya A. Alsanory
  • Tasneem A. Alsanory


Ras-related domain family member 1 transcript variant A (RASSF1A) controls apoptosis and cell proliferation while p14/ARF gene has a regulatory role in cellular senescence. Failure of apoptosis and cellular senescence occurs during inflammatory bowel disease (IBD) and colorectal cancer (CRC). To reveal the role of peripheral leukocyte promoter methylation of RASSF1A and p14/ARF in the pathogenesis of IBD subtypes and CRC we investigated the methylation state of the two genes by methylation-specific polymerase chain reaction (MSP-PCR) in 60 CRC patients, 60 patients with IBD; 27 with ulcerative colitis and 33 had Crohn’s disease and also in 30 healthy subjects. Methylated RASSF1A and p14/ARF genes were detected in 55% and 60% of CRC, while the frequency of the methylated RASSF1A and p14/ARF genes was 23.3% and 43.3% in IBD patients and 3.3% and 13.3% in the control group (P = 0.000 each). Also, the frequency of methylated RASSF1A gene was significantly higher in ulcerative colitis than in Crohn’s disease, while a non-significant frequency of methylated p14/ARF was detected between ulcerative colitis and Crohn’s disease. Furthermore, methylated RASSF1A and p14/ARF were associated with the grade of CRC but not associated with the age of patients, family history, or tumor location. Results suggest that methylated RASSF1A and p14/ARF are related to CRC and IBD pathogenesis and may be used as molecular biomarkers for early detection of CRC and IBD.


RASSF1A p14/ARF Methylation-specific PCR Inflammatory bowel disease Colorectal cancer 



alternative reading frame


cyclin-dependent kinase-2


colorectal cancer


inflammatory bowel disease


methylation-specific polymerase chain reaction


Ras-association domain family 1A.


Funding information

The study received support from grant office, Faculty of Medicine, Assiut University—grant no. R3-2015.

Compliance with Ethical Standards

The study was approved by the Faculty’s Ethics committee (ethical code, 17100666) and in concordance to Helsinki II declaration. All participants gave informed consent before the study start.

Conflict of Interest

The authors declared that they have no conflict of interest.


  1. 1.
    Vaiopoulos, A. G., Athanasoula, K. C., & Papavassiliou, A. G. (2014). Epigenetic modifications in colorectal cancer: molecular insights and therapeutic challenges. Biochim cal Biophys Acta - Mol Basis Dis, 1842(7), 971–980.Google Scholar
  2. 2.
    Perera, A.P., Sajnani, K., Dickinson, J., Eri, R., & Körner, H. (2018). NLRP3 inflammasome in colitis and colitis- associated colorectal cancer. Mamm Genome, (11-12), 817–830.Google Scholar
  3. 3.
    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.Google Scholar
  4. 4.
    Tahara, T., Shibata, T., Nakamura, M., Okubo, M., Yamashita, H., Yoshioka, D., Yonemura, J., Kmiya, Y., Ishizuka, T., Fujita, H., Nagasaka, M., Yamada, H., Hirata, I., & Arisawa, T. (2011). Host genetic factors, related to inflammatory response, influence the CpG island methylation status in colonic mucosa in ulcerative colitis. Anticancer Research, 31(3), 933–938.Google Scholar
  5. 5.
    Dziaman, T., Gackowski, D., Guz, J., Linowiecka, K., Bodnar, M., Starczak, M., Zarakowska, E., Modrzejewska, M., Szpila, A., Szpotan, J., Gawronski, M., Labejszo, A., Liebert, A., Banaszkiewicz, Z., Klopocka, M., Foksinski, M., Marszalek, A., & Olinski, R. (2018). Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions-benign adenomas and inflammatory bowel disease. Clinical Epigenetics, 10(1), 1–11.Google Scholar
  6. 6.
    Lin, Z., Hegarty, J., Cappe, J., Yu, W., Chen, X., Faber, P., Wang, Y., Kelly, A. A., Portiz, L. P., & Peterson, B. Z. (2011). Identification of disease-associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease. Clinical Genetics, 80(1), 59–67.Google Scholar
  7. 7.
    Rashid, A., Shen, L., Morris, J. S., Issa, J. P., & Hamilton, S. R. (2001). CpG island methylation in colorectal adenomas. American Journal of Pathololog, 159(3), 1129–1135.Google Scholar
  8. 8.
    Cassinotti, E., Melson, J., Liggett, T., Melnikov, A., Yi, Q., Replogle, C., Mobarhan, S., Boni, L., Segato, S., & Levenson, V. (2012). DNA methylation patterns in blood of patients with colorectal cancer and adenomatous colorectal polyps. International Journal of Cancer, 31(5), 1153–1157.Google Scholar
  9. 9.
    Donninger, H., Vos, M. D., & Clark, G. J. (2007). The RASSF1A tumor suppressor. Journal of Cell Science, 120(18), 3163–3172.Google Scholar
  10. 10.
    Ng, J. M. K., & Yu, J. (2015). Promoter hypermethylation of tumour suppressor genes as potential biomarkers in colorectal cancer. International Journal Mololecular Science, 16(2), 2472–2496.Google Scholar
  11. 11.
    Chen, T., Yang, Z., Liu, C., Wang, L., Yang, J., Chen, L., & Li, W. (2018). Circ_0078767 suppresses non-small-cell lung cancer by protecting RASSF1A expression via sponging miR-330-3p. Cell Proliferation, 3, e12548.7.Google Scholar
  12. 12.
    Balgkouranidou, I., Matthaios, D., Karayiannakis, A., Bolanaki, H., Michailidis, P., Xenidis, N., Amarantidis, K., Chelis, L., Trypsianis, G., Chatzaki, E., Lianidou, E. S., & Kakolyris, S. (2015). Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients. Mutatation Researsh, 778, 46–51.Google Scholar
  13. 13.
    Pasha, H.F., Mohamed, R. H., & Radwan, M.I. (2019). RASSF1A and SOCS1 genes methylation status as a noninvasive marker for hepatocellular carcinoma. Cancer Biomarkers, 24(2), 241–247.Google Scholar
  14. 14.
    Rezk, N. A., Mohamed, R. H., Alnemr, A. A., & Harira, M. (2018). Promoter methylation of RASSF1A gene in egyptian patients with ovarian cancer. Applied Biochemistry and Biotechnology, 185(1), 153–162.Google Scholar
  15. 15.
    Cao, X., Tang, Q., Holland-Letz, T., Gündert, M., Cuk, K., Schott, S., Heil, J., Golatta, M., Sohn, C., Schneeweiss, A. & Burwinkel, B. (2018). Evaluation of promoter methylation of RASSF1A and ATM in peripheral blood of breast cancer patients and healthy control individuals. International Journal of Molecular Science, (900), 1–12.Google Scholar
  16. 16.
    Matthaios, D., Balgkouranidou, I., Karayiannakis, A., Bolanaki, H., Xenidis, N., Amarantidis, K., Chelis, L., Romanidis, K., Chatzaki, A., Lianidou, E., Trypsianis, G., & Kakolyris, S. (2016). Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer. Oncology Letter, 12(1), 748–756.Google Scholar
  17. 17.
    Wang, Y. C., Yu, Z. H., Liu, C., Xu, L. Z., Yu, W., Lu, J., Zhu, R. M., Li, G. L., Xia, X. Y., Wei, X. W., Ji, H. Z., Lu, H., Gao, Y., Gao, W. M., & Chen, L. B. (2008). Detection of RASSF1A promoter hypermethylation in serum from gastric and colorectal adenocarcinoma patients. World Journal of Gastroenterology, 14(19), 3074–3080.Google Scholar
  18. 18.
    Rivandi, M., Khorrami, M. S., Fiuji, H., Shahidsales, S., Hasanzadeh, M., Jazayeri, M. H., Hassanian, S. M., Ferns, G. A., Saghafi, N., & Avan, A. (2018). The 9p21 locus. A potential therapeutic target and prognostic marker in breast cancer. Journal of Cellular Physiology, 233(7), 5170–5179.Google Scholar
  19. 19.
    Tadesse, S., Caldon, E., Tilley, W., & Wang, S. (2018). Cyclin dependent kinase 2 inhibitors in cancer therapy: an update. Journal of Medicinal Chemistry.Google Scholar
  20. 20.
    Mikawa, R., Suzuki, Y., Baskoro, H., Kanayama, K., Sugimoto, K., Sato, T., & Sugimoto, M. (2018). Elimination of p19ARF-expressing cells protects against pulmonary emphysema in mice. Aging Cell, 17(5), e12827.Google Scholar
  21. 21.
    Mekky, M. A., Salama, R. H., Abdel-Aal, M. F., Ghaliony, M. A., & Zaky, S. (2018). Studying the frequency of aberrant DNA methylation of APC, P14, and E-cadherin genes in HCV-related hepatocarcinogenesis. Cancer Biomark, 22(3), 503–509.Google Scholar
  22. 22.
    Hibi, K., Nakayama, H., Koike, M., Kasai, Y., Ito, K., Akiyama, S., & Nakao, A. (2002). Colorectal cancers with both p16 and p14 methylation show invasive characteristics. Jpn J Cancer Res, 93(8), 883–887.Google Scholar
  23. 23.
    Kim, S. H., Park, K. H., Shin, S. J., Lee, K. Y., Kim, T. I., Kim, N. K., Rha, S. Y., & Ahn, J. B. (2018). CpG island methylator phenotype and methylation of Wnt pathway genes together predict survival in patients with colorectal cancer. Yonsei Medical Journal, 59(5), 588–594.Google Scholar
  24. 24.
    Chaar, I., Amara, S., Elamine, O. E., Khiari, M., Ounissi, D., Khalfallah, T., Ben Hmida, A., Mzabi, S., & Bouraoui, S. (2014). Biological significance of promoter hypermethylation of p14/ARF gene: relationships to p53 mutational status in Tunisian population with colorectal carcinoma. Tumour Biology, 35(2), 1439–1449.Google Scholar
  25. 25.
    Shen, L., Catalano, P. J., Benson, A. B., O'Dwyer, P., Hamilton, S. R., & Issa, J. P. (2007). Association between DNA methylation and shortened survival in patients with advanced colorectal cancer treated with 5-fluorouracil based chemotherapy. Clinical Cancer Research, 13(20), 6093–6098.Google Scholar
  26. 26.
    Das, V., Kalita, J., & Pal, M. (2017). Predictive and prognostic biomarkers in colorectal cancer: systematic review of recent advances and challenges. Biomedicine & Pharmacotherapy, 87, 8–19.Google Scholar
  27. 27.
    Farahzadi, R., Fathi, E., Mesbah-Namin, S. A., & Zarghami, N. (2017). Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells. PLoS ONE, 12(11), e0188052.Google Scholar
  28. 28.
    Lorente, A., Mueller, W., Urdangarín, E., Lázcoz, P., von Deimling, A., & Castresana, J. S. (2008). Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR. BMC Cancer, 8(1), 61.Google Scholar
  29. 29.
    Farahzadi, R., Fathi, E., Mesbah-Namin, S.A., & Zarghami, N. (2018). Anti-aging protective effect of L-carnitine as clinical agent in regenerative medicine through increasing telomerase activity and change in the hTERT promoter CpG island methylation status of adipose tissue-derived mesenchymal stem cells. Tissue and Cel1, 54, 105–113.Google Scholar
  30. 30.
    Ashktorab, H., & Brim, H. (2015). DNA methylation and colorectal cancer. Current Colorectal Cancer Reports, 10(4), 425–430.Google Scholar
  31. 31.
    Yoon, J. H., Dammann, R., & Pfeifer, G. P. (2001). Hypermethylation of the CpG island of the RASSF1A gene in ovarian and renal cell carcinomas. International Journal Cancer, 4, 9212–9217.Google Scholar
  32. 32.
    Wagner, K. J., Cooper, W. N., Grundy, R. G., Caldwell, G., Jones, C., Wadey, R. B., Morton, D., Schofield, P. N., Reik, W., Latif, F., & Maher, E. R. (2000). Frequent RASSF1A tumor suppressor gene promoter methylation in Wilms’ tumor and colorectal cancer. Oncogene, 21, 7277–7282.Google Scholar
  33. 33.
    Karatzas, P. S., Gazouli, M., Safioleas, M., & Mantzaris, G. J. (2014). DNA methylation changes in inflammatory bowel disease. Annals of Gastroenterology, 27, 125–132.Google Scholar
  34. 34.
    Abouzeid, H. E., Kassem, A. M., Wahab Abdel, A. H., El-mezayen, H. A., Sharad, H., & Abdel Rahman, S. (2011). Promoter hypermethylation of RASSF1A, MGMT, and HIC-1 genes in benign and malignant colorectal tumors. Tumor Biology, 32, 845–852.Google Scholar
  35. 35.
    Zheng, W. W., Zhao, L. D., Wang, G. X., Kang, X. C., Qin, L., Ji, J. J., & Hao, S. (2016). Promoter methylation and expression of RASSF1A genes as predictors of disease progression in colorectal cancer. International Journal of Clinical and Experimental Medicineis, 9(2), 2027–2036.Google Scholar
  36. 36.
    van Engeland, M., Roemen, G. M., Brink, M., Pachen, M. M., Weijenberg, M. P., de Bruïne, A. P., Arends, J. W., van den Brandt, P. A., de Goeij, A. F., & Herman, J. G. (2002). K-ras mutations and RASSF1A promoter methylation in colorectal cancer. Oncogene, 21(23), 3792–3795.Google Scholar
  37. 37.
    Cleven, A. H., Derks, S., Draht, M. X., Smits, K. M., Melotte, V., Van Neste, L., Tournier, B., Jooste, V., Chapusot, C., Weijenberg, M. P., et al. (2014). CHFR promoter methylation indicates poor prognosis in stage II microsatellite stable colorectal cancer. Clinical Cancer Research, 20(12), 3261–3271.Google Scholar
  38. 38.
    Nilsson, T. K., Lof-Ohlin, Z. M., & Sun, X. F. (2013). DNA methylation of the p14ARF, RASSF1A and APC1A genes as an independent prognostic factor in colorectal cancer patients. International Journal Oncololy, 42(1), 127–133.Google Scholar
  39. 39.
    Li, M., Yan, D. G., & Liu, J. L. (2016). Methylation status of PCDH10 and RASSF1A gene promoters in colorectal cancer. Zhonghua Yi Xue Za Zh, 96(6), 456–459.Google Scholar
  40. 40.
    Hu, H., Zhou, C., Li, B., Chen, Y., Dai, J., Mao, Y., Huang, T., Yu, H., Chen, M., Zhao, J., & Duan, S. (2018). Diagnostic value of RASSF1A hypermethylation in colorectal cancer: a meta-analysis. Pathology, Research and Practice, 214(10), 1572–1578.Google Scholar
  41. 41.
    Chen, S. P., Wu, C. C., Huang, S. Y., Kang, J. C., Chiu, S. C., Yang, K. L., & Pang, C. Y. (2012). Beta-catenin and K-ras mutations and RASSF1A promoter methylation in Taiwanese colorectal cancer patients. Genetic Testing and Moleclar Biomarkers, 16(11), 1277–1281.Google Scholar
  42. 42.
    An, B., Kondo, Y., Okamoto, Y., Shinjo, K., Kanemitsu, Y., Komori, K., Hirai, T., Sawaki, A., Tajika, M., Nakamura, T., Yamao, K., Yatabe, Y., Fujii, M., Murakami, H., Osada, H., Tani, T., Matsuo, K., Shen, L., Issa, J. P., & Sekido, Y. (2010). Characteristic methylation profile in CpG island methylator phenotype-negative distal colorectal cancers. International Journal of Cancer, 127(9), 2095–2105.Google Scholar
  43. 43.
    Gordon, M., El-Kalla, M., Zhao, Y., Fiteih, Y., Law, J., Volodko, N., Anwar-Mohamed, A., El-Kadi, A. O., Liu, L., Odenbach, J., Thiesen, A., Onyskiw, C., Ghazaleh, H. A., Park, J., Lee, S. B., Yu, V. C., Fernandez-Patron, C., Alexander, R. T., Wine, E., & Baksh, S. (2013). The tumor suppressor gene, RASSF1A, is essential for protection against inflammation -induced injury. PLoS One, 8(10), e75483.Google Scholar
  44. 44.
    Nterma, P., Panopoulou, E., Papadaki-Petrou, E., & Assimakopoulou, M. (2019). Immunohistochemical profile of tumor suppressor proteins RASSF1A and LATS1/2 in relation to p73 and YAP Expression of human inflammatory bowel disease and normal intestine. Pathology Oncolology Research.
  45. 45.
    Sato, F., Harpaz, N., Shibata, D., Xu, Y., Yin, J., Mori, Y., Zou, T., Wang, S., Desai, K., Leytin, A., Selaru, F. M., Abraham, J. M., & Meltzer, S. J. (2002). Hypermethylation of the p14 ARF gene in ulcerative colitis-associated colorectal carcinogenesis. Cancer Research, 62(4), 1148–1151.Google Scholar
  46. 46.
    Kang, M. Y., Lee, B. B., Ji, Y. I., Jung, E. H., Chun, H. K., Song, S. Y., Park, S. E., Park, J., & Kim, D. H. (2008). Association of interindividual differences in p14ARF promoter methylation with single nucleotide polymorphism in primary colorectal cancer. Cancer, 112(8), 1699–1707.Google Scholar
  47. 47.
    Moriyama, T., Matsumoto, T., Nakamura, S. Y., Mibu, R., Yao, T., & Iida, M. (2007). Hypermethylation of p14 (ARF) may be predictive of colitic cancer in patients with ulcerative colitis. Diseases of the Colon and Rectum, 50(9), 1384–1392.Google Scholar
  48. 48.
    Garrity-Park, M. M., Loftus, E. V., Sandborn, W. J., Brayant, S., & Symrck, T. (2010). Methylation status of genes in non-neoplastic mucosa from patients with ulcerative colitis-associated colorectal cancer. American Journal of Gastroenterology, 105(7), 1610–1619.Google Scholar
  49. 49.
    Esteller, M., Tortola, S., Toyota, M., Capella, G., Peinado, M. A., Baylin, S. B., & Herman, J. G. (2000). Hypermethylation-associated inactivation of p14(ARF) is independent of p16 (INK4a) methylation and p53 mutational status. Cancer Research, 60(1), 129–133.Google Scholar
  50. 50.
    Burri, N., Shaw, P., Bouzourene, H., Sordat, I., Sordat, B., Gillet, M., Schorderet, D., Bosman, F. T., & Chaubert, P. (2001). Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer. Labatory Investigation, 81(2), 217–229.Google Scholar
  51. 51.
    Naghibalhossaini, F., Hosseini, H. M., Mokarram, P., & Zamani, M. (2011). High frequency of genes’ promoter methylation, but lack of BRAF V600E mutation among Iranian colorectal cancer patients. Pathology & Oncology Research, 17(4), 819–825.Google Scholar
  52. 52.
    Kim, J. C., Choi, J. S., Roh, S. A., Cho, D. H., Kim, T. W., & Kim, Y. S. (2010). Promoter methylation of specific genes is associated with the phenotype and progression of colorectal adenocarcinomas. Annals of Surgical Oncology, 17(7), 1767–1776.Google Scholar
  53. 53.
    Lind, G. E., Thorstensen, L., Løvig, T., Meling, G. I., Hamelin, R., Rognum, T. O., et al. (2004). A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines. Molecular Cancer, 3(1), 28.Google Scholar
  54. 54.
    Zheng, S., Chen, P., McMillan, A., Lafuente, A., Lafuente, M. J., Ballesta, A., Trias, M., & Wiencke, J. K. (2000). Correlations of partial and extensive methylation at the p14ARF locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors. Carcinogenesis, 21(11), 2057–2064.Google Scholar
  55. 55.
    Zhou, Z., Zhang, H., Lai, J., Diao, D., Li, W., Dang, C., & Song, Y. (2016). Relationships between p14ARF gene methylation and clinicopathological features of colorectal cancer: a meta-analysis. Colorectal cancer: a meta-analysis. PLoS ONE, 11(3), 1–12.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ragaa H. Salama
    • 1
  • Zain El-Abdeen A. Sayed
    • 2
  • Ahmed M. Ashmawy
    • 2
  • Wael A. Elsewify
    • 3
  • Ghada M. Ezzat
    • 1
    Email author
  • Mahmoud A. Mahmoud
    • 2
  • Aya A. Alsanory
    • 4
  • Tasneem A. Alsanory
    • 4
  1. 1.Department of Medical Biochemistry, Faculty of MedicineAssiut UniversityAssiutEgypt
  2. 2.Department of Internal Medicine, Faculty of MedicineAssiut UniversityAssiutEgypt
  3. 3.Department of Internal Medicine, Faculty of MedicineAswan UniversityAswanEgypt
  4. 4.Students at Faculty of Medicine, Faculty of PharmacyAssiut UniversityAssiutEgypt

Personalised recommendations