Comparison of the anti-cancer effect of Disulfiram and 5-Aza-CdR on pancreatic cancer cell line PANC-1


Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Pancreatic cancer has poor prognosis by surgical and chemotherapy when it is diagnosed, so other anti-cancerous assistant therapeutic drugs are suggested e.g. epigenetic reversal of tumor-suppressor genes on promoter hypermethylation. 5-Aza-CdR is a nucleoside analog of DNMTi but it has long-term cytotoxicity effects. This study compares the anticancer effect of 5-Aza-CdR and Disulfiram potencies on PANC-1 cell line and up-regulation of p21.
Materials and Methods : PANC-1 cell line was cultured in DMEM high glucose and treated by 5-Aza-CdR with 5 and 10 μM concentration for four days and 13 μM DSF (Diulfiram) for 24 hours. MS-PCR and RT-PCR were carried out to detect the methylation pattern and estimate the mRNA expression of RASSF1A and p21 in PANC-1.
Result : MS-PCR demonstrated partial unmethylation after treatment with 5-Aza-CdR while there was no unmethylated band after DSF treatment. RT-PCR showed significant differences between re-expression of RASSF1A before and after treatment with 10 μM 5-Aza-CdR (P < 0.01) but not after treatment with 13 μM DSF (P > 0.05). The significant correlation was observed between RASSF1A re-expression and p21 up-regulation before and after treatment with 10 μM 5-Aza-CdR (P < 0.01) but not after treatment with 13 μM DSF (P > 0.05), while p21 up-regulation was significantly higher after DSF treatment (P < 0.01).
Conclusion: Our findings indicated that 5-Aza-CdR induces the re-expression of RASSF1A and p21 up-regulation in PANC-1. DSF showed no epigenetic reversion while it affected p21 up-regulation.


1. Hariharan D, Saied A, Kocher HM. Analysis of mortality rates for pancreatic cancer across the world. HPB (Oxford) 2008;10:58-62.  Back to cited text no. 1
2. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al. Identification of pancreatic cancer stem cells. Cancer Res 2007;67:1030-7.  Back to cited text no. 2
3. Hruban RH, Brune K, Fukushima N, Maitra A. Pancreatic intraepithelial neoplasia. Pancreatic Cancer. 2008;378:41-51  Back to cited text no. 3
4. Dammann R, Schagdarsurengin U, Liu L, Otto N, Gimm O, Dralle H, et al. Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma. Oncogene 2003;22:3806-12.  Back to cited text no. 4
5. Arlt A, Müerköster SS, Schäfer H. Targeting apoptosis pathways in pancreatic cancer. Cancer Lett 2013;332:346-58.  Back to cited text no. 5
6. Cohen Y, Singer G, Lavie O, Dong SM, Beller U, Sidransky D. The RASSF1A tumor suppressor gene is commonly inactivated in adenocarcinoma of the uterine cervix. Clin Cancer Res 2003;9:2981-4.  Back to cited text no. 6
7. Abouzeid HE, Kassem AM, Abdel Wahab AH, El-mezayen HA, Sharad H, Abdel Rahman S. Promoter hypermethylation of RASSF1A, MGMT, and HIC-1 genes in benign and malignant colorectal tumors. Tumour Biol 2011;32:845-52.  Back to cited text no. 7
8. Fandy TE. Development of DNA methyltransferase inhibitors for the treatment of neoplastic diseases. Curr Med Chem 2009;16:2075-85.  Back to cited text no. 8
9. Cortez CC, Jones PA. Chromatin, cancer and drug therapies. Mutat Res 2008;647:44-51.  Back to cited text no. 9
10. Esteller M. Epigenetics in cancer. N Engl J Med 2008;358:1148-59.  Back to cited text no. 10
11. Momparler RL. Cancer epigenetics. Oncogene 2003;22:6479-83.  Back to cited text no. 11
12. Billam M, Sobolewski MD, Davidson NE. Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells. Breast Cancer Res Treat 2010;120:581-92.  Back to cited text no. 12
13. Barth KS, Malcolm RJ. Disulfiram: An old therapeutic with new applications. CNS Neurol Disord Drug Targets 2010;9:5-12.  Back to cited text no. 13
14. Iljin K, Ketola K, Vainio P, Halonen P, Kohonen P, Fey V, et al. High-throughput cell-based screening of 4910 known drugs and drug-like small molecules identifies disulfiram as an inhibitor of prostate cancer cell growth. Clin Cancer Res 2009;15:6070-8.  Back to cited text no. 14
15. Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor. J Cell Sci 2007;120:3163-72.  Back to cited text no. 15
16. Shivakumar L, Minna J, Sakamaki T, Pestell R, White MA. The RASSF1A tumor suppressor blocks cell cycle progression and inhibits cyclin D1 accumulation. Mol Cell Biol 2002;22:4309-18.  Back to cited text no. 16
17. Sherwood V, Recino A, Jeffries A, Ward A, Chalmers AD. The N-terminal RASSF family: A new group of Ras-association-domain-containing proteins, with emerging links to cancer formation. Biochem J 2009;425:303-11.  Back to cited text no. 17
18. Kierszenbaum AL. Histology and Cell Biology: An Introduction to Pathology. E-Book. 2 nd ed.; 2007.  Back to cited text no. 18
19. Thaler S, Hähnel PS, Schad A, Dammann R, Schuler M. RASSF1A mediates p21Cip1/Waf1-dependent cell cycle arrest and senescence through modulation of the Raf-MEK-ERK pathway and inhibition of Akt. Cancer Res 2009;69:1748-57.  Back to cited text no. 19
20. Stresemann C, Brueckner B, Musch T, Stopper H, Lyko F. Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines. Cancer Res 2006;66:2794-800.  Back to cited text no. 20
21. Lin J, Haffner MC, Zhang Y, Lee BH, Brennen WN, Britton J, et al. Disulfiram is a DNA demethylating agent and inhibits prostate cancer cell growth. Prostate 2011;71:333-43.  Back to cited text no. 21
22. Avramouli A, Tsochas S, Mandala E, Katodritou E, Ioannou M, Ritis K, et al. Methylation status of RASSF1A in patients with chronic myeloid leukemia. Leuk Res 2009;33:1130-2.  Back to cited text no. 22
23. Yang B, House MG, Guo M, Herman JG, Clark DP. Promoter methylation profiles of tumor suppressor genes in intrahepatic and extrahepatic cholangiocarcinoma. Mod Pathol 2005;18:412-20.  Back to cited text no. 23
24. Schagdarsurengin U, Wilkens L, Steinemann D, Flemming P, Kreipe HH, Pfeifer GP, et al. Frequent epigenetic inactivation of the RASSF1A gene in hepatocellular carcinoma. Oncogene 2003;22:1866-71.  Back to cited text no. 24
25. Vos MD, Ellis CA, Bell A, Birrer MJ, Clark GJ. Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis. Science's STKE 2000;275:35669.  Back to cited text no. 25
26. Lund AH, van Lohuizen M. Epigenetics and cancer. Genes Dev 2004;18:2315-35.  Back to cited text no. 26
27. Shen WJ, Dai DQ, Teng Y, Liu HB. Regulation of demethylation and re-expression of RASSF1A gene in gastric cancer cell lines by combined treatment of 5-Aza-CdR and NaB. World J Gastroenterol 2008;14:595-600.  Back to cited text no. 27
28. Harada K, Toyooka S, Maitra A, Maruyama R, Toyooka KO, Timmons CF, et al. Aberrant promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines. Oncogene 2002;21:4345-9.  Back to cited text no. 28
29. Brueckner B, Kuck D, Lyko F. DNA methyltransferase inhibitors for cancer therapy. Cancer J 2007;13:17-22.  Back to cited text no. 29
30. Qiu H, Yashiro M, Shinto O, Matsuzaki T, Hirakawa K. DNA methyltransferase inhibitor 5-aza-CdR enhances the radiosensitivity of gastric cancer cells. Cancer Sci 2009;100:181-8.  Back to cited text no. 30
31. Chiam K, Centenera MM, Butler LM, Tilley WD, Bianco-Miotto T. GSTP1 DNA methylation and expression status is indicative of 5-aza-2'-deoxycytidine efficacy in human prostate cancer cells. PLoS One 2011;6:e25634.  Back to cited text no. 31
32. Agathanggelou A, Cooper WN, Latif F. Role of the Ras-association domain family 1 tumor suppressor gene in human cancers. Cancer Res 2005;65:3497-508.  Back to cited text no. 32
33. Zuo S, Chen Y, Xu L, Tang Q, Zou S. Re-expression of RASSF1A by 5-Aza-CdR induced demethylation of the promoter region in human biliary tract carcinoma cells. J Huazhong Univ Sci Technolog Med Sci 2007;27:281-4.  Back to cited text no. 33
34. Yu JN, Xue CY, Wang XG, Lin F, Liu CY, Lu FZ, et al. 5-AZA-2'-deoxycytidine (5-AZA-CdR) leads to down-regulation of Dnmt1o and gene expression in preimplantation mouse embryos. Zygote 2009;17:137-45.  Back to cited text no. 34
35. Christman JK. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: Mechanistic studies and their implications for cancer therapy. Oncogene 2002;21:5483-95.  Back to cited text no. 35
36. Rui L, Xue WJ, Li P, Wang ZW, Wang P, Li HX. [Inhibitory effect of wild-type RASSF1A gene expression on proliferation of hepatocellular carcinoma QGY-7703 cells]. Ai Zheng 2008;27:924-8.  Back to cited text no. 36
37. Lyko F, Brown R. DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J Natl Cancer Inst 2005;97:1498-506.  Back to cited text no. 37
38. Lin X, Asgari K, Putzi MJ, Gage WR, Yu X, Cornblatt BS, et al. Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide. Cancer Res 2001;61:8611-6.  Back to cited text no. 38
39. Lloyd AC, Obermüller F, Staddon S, Barth CF, McMahon M, Land H. Cooperating oncogenes converge to regulate cyclin/cdk complexes. Genes Dev 1997;11:663-77.  Back to cited text no. 39
40. Roper E, Weinberg W, Watt FM, Land H. p19ARF-independent induction of p53 and cell cycle arrest by Raf in murine keratinocytes. EMBO Rep 2001;2:145-50.  Back to cited text no. 40
41. Sewing A, Wiseman B, Lloyd AC, Land H. High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1. Mol Cell Biol 1997;17:5588-97.  Back to cited text no. 41
42. Wickström M, Danielsson K, Rickardson L, Gullbo J, Nygren P, Isaksson A, et al. Pharmacological profiling of disulfiram using human tumor cell lines and human tumor cells from patients. Biochem Pharmacol 2007;73:25-33.  Back to cited text no. 42
43. Chen D, Cui QC, Yang H, Dou QP. Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and xenografts via inhibition of the proteasome activity. Cancer Res 2006;66:10425-33.  Back to cited text no. 43
44. Wang W, McLeod HL, Cassidy J. Disulfiram-mediated inhibition of NF-kappaB activity enhances cytotoxicity of 5-fluorouracil in human colorectal cancer cell lines. Int J Cancer 2003;104:504-11.  Back to cited text no. 44
45. Cho HJ, Lee TS, Park JB, Park KK, Choe JY, Sin DI, et al. Disulfiram suppresses invasive ability of osteosarcoma cells via the inhibition of MMP-2 and MMP-9 expression. J Biochem Mol Biol 2007;40:1069-76.  Back to cited text no. 45